Inhibition of Lysophosphatidate Signaling by Lidocaine and Bupivacaine 

The study protocol was approved by the Animal Research Committee of the University of Virginia. Adult female Xenopus laevis frogs were obtained from Xenopus I (Ann Arbor, MI) and housed in an established frog colony. To obtain oocytes, frogs were anesthetized with 1% tricaine (3-aminobenzoic acid ethyl ester) until they became unresponsive, and they were operated on ice. Oocytes were harvested through a 5-mm incision in the lower lateral abdominal wall and immediately placed in modified Barth's solution (containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO³, 0.41 mM CaCl², 0.82 mM MgSO⁴, 0.3 mM Ca(NO³)², 15 mM HEPES, and 0.1 mM mg/ml gentamicin, with pH adjusted to 7.6). Frogs were returned to the main tank after recovering for 2 days in isolation. Oocytes (Dumont's stage 5 and 6) were defolliculated manually using microforceps and incubated in modified Barth's solution at 18 degrees Celsius for 24 h because this results in more consistent lysophosphatidate responses.

Endogenous lysophosphatidate receptors were used to evaluate the influence of local anesthetics on lysophosphatidate signaling. The angiotensin receptor was expressed recombinantly. A 1.2-kilobase pair cDNA in the CDM8 vector (Invitrogen, San Diego, CA), encoding the rat AT sub 1A angiotensin II receptor, was a gift from Dr. K. R. Lynch, of the University of Virginia. The construct was linearized with the nuclease Xho I and transcribed in vitro by T7 RNA polymerase in the presence of a capping analog. Oocytes were injected with 50 nl mRNA (5 ng) in water, using an automated microinjector (Nanoject; Drummond Scientific, Broomall, PA). Slight ballooning of the oocyte confirmed the adequacy of injection. The cells were cultured for 72 h at 18 degrees Celsius in modified Barth's solution before study.

Lysophosphatidate or angiotensin receptor activation in the oocyte leads to G protein-induced phospholipase C activity and release of IP³from phosphoinositolbisphosphate (Figure 1(A)). Inositoltrisphosphate then binds to its specific receptor on intracellular Ca²+ stores to release Ca²+, which in turn opens an endogenous Ca²+-activated Cl sup - channel. The resulting current can be measured using a voltage clamp, and this Ca²+-activated Cl sup - current (I^Cl(Ca)), integrated and expressed in microcoulombs (micro Coulomb), is a measure of intracellular Ca²+ release. [8,12–14] All experiments were performed at room temperature (approximately 22 degrees Celsius).

Microelectrodes were pulled in one stage from capillary glass (BBL with fiber; World Precision Instruments, Sarasota, FL) on a micropipette puller (model 700C; David Kopf Instruments, Tujunga, CA). Tips were broken to a diameter of approximately 10 mM, providing a resistance of 1–3 M Ohm, and filled with 3 M KCl. A single oocyte was placed in a perfusable bath containing 3 ml Tyrode's solution (consisting of 150 mM NaCl, 5 mM KCl, 1 mM MgCl², 2 mM CaCl², 10 mM dextrose, and 10 mM HEPES, with the p3H adjusted to 6.4 with NaOH) with or without local anesthetic. Two microelectrodes were inserted into the oocyte, and a holding potential of -70 mV was applied to the membrane. The voltage clamp amplifier (OC725A; Warner Corp., New Haven, CT) was connected to a data acquisition and analysis system running on an IBM-compatible personal computer. The acquisition system was a DAS-8 A/D conversion board (Keithly-Metrabyte, Taunton, MA), and analysis was performed with OoClamp software. [15] Occasional cells that did not show a stable holding current less than 1 mA during a 1-min equilibration period were excluded from analysis. Membrane current was sampled at 125 Hz and recorded for 5 s before and 55 s after application of the agonist (lysophosphatidate or angiotensin, 10 sup -7 M). Agonists were delivered in 30-ml aliquots during 1 or 2 s using a micropipettor positioned approximately 1 mm from the oocyte. Responses were quantified by integrating the current trace by quadrature and are reported as microcoulombs because this reflects intracellular Ca²+ release better than peak current does. [16] Oocytes were preincubated with local anesthetics at least 2 min before agonists were applied. In pilot experiments we found that inhibition develops within 60 s after exposure to the local anesthetic.

For intracellular injections of IP³, a third microelectrode connected to an automated microinjector (the Nanoject) was inserted into the oocyte. Cells were injected under voltage clamp, and the adequacy of injection was verified by observing the small increase in cell size on injection.

Results are reported as means +/- standard error of the mean. Because variability among batches of oocytes is common, responses were at times normalized to controls for each batch. Differences between treatment groups were analyzed using analysis of variance and Student's unpaired t test. Probability values less than 0.05 were considered significant.

Molecular biology reagents were obtained from Promega (Madison, WI), and other chemicals were obtained from Sigma Chemical Company (St. Louis, MO). Lidocaine HCl and bupivacaine HCl (Sigma Chemical Co.) were diluted in Tyrode solution. Because of bupivacaine's low solubility at pH values greater than 6.4, all solutions were adjusted to pH of 6.4, and pilot experiments showed that lysophosphatidate and angiotensin signaling were not influenced by this lower value. Consecutive dilutions for dose-response calculations were made with pH-adjusted Tyrode solution. Lysophosphatidate (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate [sodium salt]; Avanti Polar Lipids, Alabaster, AL) was prepared as a 10 sup -4 M stock solution in 1% delipidated bovine serum albumin (ICN Biomedicals, Aurora, OH). Consecutive dilutions to 10 sup -7 M were made in Tyrode solution. Before experiments, lysophosphatidate solutions were sonicated for 5 s using a Branson Sonifier 250 (Misonix, Farmingdale, NY) to ensure that the compound was fully in solution. All other chemicals were obtained from Sigma Chemical Company.

Oocytes responded to 30 ml 10 sup -7 M lysophosphatidate with a transient inward current (Figure 1(B)). The vehicle bovine serum albumin had no effect. The current developed after 1–3 s and consisted of a fast component and a gradual return to baseline in 10 to 20 s, which is the typical response to G protein-coupled receptor stimulation in Xenopus oocytes. The average control response was 2.4 +/- 0.2 micro Coulomb.

Lysophosphatidate signaling was inhibited when oocytes were exposed to lidocaine or bupivacaine at concentrations used clinically for injection around wounds (Figure 2(A)). The inhibitory effect was concentration dependent; the IC⁵⁰was 29.9 +/- 8.2 mM for lidocaine (Figure 2(B)) and 4.5 +/- 1.2 mM for bupivacaine (Figure 2(C)). Hill coefficients were 1.5 and 1.6, respectively. At high local anesthetic concentrations, virtually complete inhibition of lysophosphatidate signaling was observed. In the presence of local anesthetics, a modest but consistent slowing of the response was noted, but we did not investigate the mechanism any further.

The reversibility of the local anesthetic inhibitory effect on lysophosphatidate signaling was investigated in three separate groups of oocytes because we were concerned that multiple lysophosphatidate applications might desensitize the lysophosphatidate receptor (Figure 3). In the first group, responses to 10 sup -7 M lysophosphatidate were recorded in Tyrode solution. In the second group, responses were determined in the presence of 42.8 mM lidocaine (1%) or 15.4 mM bupivacaine (0.5%); inhibitions to 26 +/- 7% and 11 +/- 3%, respectively, of control values were seen. In the third group, oocytes were first placed in 42.8 mM lidocaine or 15.4 mM bupivacaine for 5 min, which was then completely washed out with Tyrode solution. After 5 min of washing, responses to lysophosphatidate were recorded. Responses after bupivacaine washout were 115 +/- 17% of control, and thus inhibition by bupivacaine was completely reversible. Responses after lidocaine washout were 58 +/- 11% of control. Although this was not significantly different from control values, it suggests a prolonged and possibly irreversible effect of lidocaine.

To determine that the effect of lidocaine and bupivacaine on lysophosphatidate signaling was specific and not due to nonspecific membrane interactions or inhibition of the Cl sup - channel, we expressed the angiotensin^1Areceptor, which like the lysophosphatidate receptor is a member of the G protein-coupled receptor superfamily. The angiotensin^1Areceptor, when expressed in oocytes, also induces I^Cl(Ca) through a mechanism that, apart from receptor and possibly G protein, is considered to be the same as that for lysophosphatidate signaling. Lack of local anesthetic inhibition on angiotensin signaling would make unlikely a nonspecific membrane effect or an effect on the signaling pathway downstream of the lysophosphatidate receptor (including the Cl sup - channel). Angiotensin II (10 sup -7 M) induced no responses in uninjected oocytes (data not shown) but did induce currents in angiotensin^1A-injected oocytes that were indistinguishable from those induced by lysophosphatidate (Figure 4(A); the average control response was 1.6 micro Coulomb). Lidocaine and bupivacaine were without effect on angiotensin signaling. I^Cl(Ca) in response to 10 sup -7 M angiotensin were 111.1 +/- 22% of control in the presence of lidocaine (42.7 mM) and 92.1 +/- 38.1% of control in the presence of bupivacaine (15.5 mM)(Figure 4). Therefore bupivacaine and lidocaine do not appear to affect the oocyte membrane, intracellular pathways, or the Ca²+-activated Cl sup - channel.

To confirm that local anesthetics do not interfere with intracellular Ca²+ release or the Ca²+-activated Cl sup - channel, we determined the effects of lidocaine and bupivacaine on I^Cl(Ca) induced by microinjection of IP³into the oocyte. Previously we showed that heparin, an IP³receptor antagonist, inhibits lysophosphatidate-induced I^Cl(Ca) completely, and thus that lysophosphatidate-induced Ca²+ release is mediated solely by IP³. [6,12,14]

Intracellular injection of 50 nl of 100 mM IP³generated I^Cl(Ca) s in the oocyte indistinguishable from those induced by extracellular lysophosphatidate (Figure 5(A)). However, in contrast to currents induced by lysophosphatidate application, size and kinetics of these responses were not affected by the presence of extracellular local anesthetic. Average responses to lysophosphatidate were 3.5 +/- 0.9 micro Coulomb in the control group, 3.3 +/- 0.8 micro Coulomb in the presence of 42.7 mM lidocaine, and 3.0 +/- 0.6 micro Coulomb in the presence of 15.5 mM bupivacaine (Figure 5(B)). These findings confirm that lysophosphatidate signaling inhibition by local anesthetics occurs early in the signaling pathway.

Our data show that the local anesthetics lidocaine and bupivacaine inhibit lysophosphatidate signaling in a concentration-dependent manner, with appreciable inhibition occurring at concentrations similar to those observed after injection around wounds. Lack of effect on signaling by angiotensin or microinjected IP³suggests that the lysophosphatidate receptor environment (receptor molecule, its lipid environment, or coupled G protein) is the site of action. These findings indicate that local anesthetics can interact significantly with lipid mediator receptors. More specifically, they may explain some observed effects of local anesthetics on the wound healing process. As bupivacaine is, and ropivacaine likely will be, used commonly to provide pain relief after surgery, the interactions of these long-acting local anesthetics with wound healing deserve to be studied in detail.

Several potential limitations of our model should be considered. First, we have only studied a single form of lysophosphatidate signaling (Cl sup - currents induced by intracellular Ca²+ release), whereas several intracellular signaling cascades are activated by lysophosphatidate: decreases in cAMP, activation of Rho, prostaglandin synthesis, and ras activation, to mention just a few. No data suggest that these effects are transduced through different subtypes of receptor. Instead they are transduced through several G proteins, presumably coupling to a single, as yet uncloned, receptor subtype. [6] Given the importance of intracellular Ca²+ signaling, we chose phospholipase C activation and IP³release as a pathway to investigate. Second, we used Xenopus oocytes to study this pathway, and consequently we performed our experiments at room temperature. This raises the question if the Xenopus lysophosphatidate receptor might behave differently from its mammalian counterpart. However, there is no molecular biological or pharmacologic evidence to suggest significant functional differences between lysophosphatidate receptors in various tissues. We have shown that the oocyte model closely replicates findings in other systems. [11,14,16] The angiotensin^1Areceptor derives from rat and therefore was expressed at a lower temperature than it normally functions in. However, this has not been shown to affect its signaling properties appreciably. Temperature differences in oocytes primarily affect latency of response of expressed receptors. [17] Therefore we believe that the flexibility of the oocyte model outweighs these minor disadvantages.

The role of lysophosphatidate in wound healing is not known. However, because the compound is released primarily from activated platelets [18] and injured fibroblasts, [5] its presence in appreciable quantities around wounds seems likely. We have found in preliminary experiments that lysophosphatidate is absent in healthy skin but released in significant quantities after injury. Because its most prominent effects are fibroblast [9,19] and smooth muscle cell [20] proliferation, vasoconstriction, [21] platelet activation, [22,23] and induction of changes in cell motility, [24–26] it seems unlikely that its presence in wounds would have no effect on the wound healing process. Therefore we postulate that lysophosphatidate plays a physiologic role in wound healing, and that interference of local anesthetics with lysophosphatidate signaling can adversely affect the healing process.

Adverse effects of local anesthetics on wound healing have been well described, [1–3,27] but the mechanisms remain unclear. Although local anesthetics are present in the wound for a short time only, their effects are noticeable for longer periods. For example, fibroblast proliferation and collagen deposition are impaired after application of local anesthetics [3] and might explain subsequent decreases in wound breaking strength. [1] Although collagen deposition does not occur until 3–5 days after wounding, [28] a time when the local anesthetic is unlikely to be present still in appreciable concentrations, local anesthetic-induced delays in fibroblast proliferation could explain this. Transient inhibition of lysophosphatidate signaling in the early stages of wound healing may disturb the subsequent wound healing cascade. Alternatively, an irreversible component of lidocaine block of lysophosphatidate signaling, as suggested by our findings, might play a role. Clinically, signs of inflammation and occasional necrosis have been observed after local anesthetic were administered around wounds. [1,27–29] Inhibition of proliferation and motility of immune system cells by local anesthetic block of lysophosphatidate signaling might explain some of these findings. On the other hand, inhibition of lysophosphatidate signaling by local anesthetics might have beneficial effects as well. Lysophosphatidate-induced vasoconstriction and platelet aggregation may be important immediately after wounding but will decrease blood flow in the regenerating wound bed. Local anesthetics might reverse this and thereby provide a better healing environment. Indeed local anesthetics have been shown to inhibit platelet aggregation, [30–32] and reverse vasospasm, [33–36] by mechanisms that have not been identified yet.

The exact site of interaction between the local anesthetics and the lysophosphatidate signaling pathway is important. Although the main site of action of local anesthetics is the Na sup + channel, [37] secondary activities exist. For example, local anesthetics inhibit Ca²+ entry through voltage-gated channels, by direct interference with the channel molecule. [38] In our study, we could rule out an effect of the anesthetics on the Ca²+-activated Cl sup - channel. As G protein-coupled pathways signal over many more intermediates than channels do, localization of the site of anesthetic action is relevant. The lack of effect on lysophosphatidate signaling after intracellular microinjection of IP³suggests that the site of action is extracellular. This is confirmed by the lack of local anesthetic effect on angiotensin signaling. The latter finding, in addition, makes a nonspecific membrane effect unlikely. Therefore, the most likely site of action is the lysophosphatidate receptor itself. The lipid moiety of the local anesthetic molecule might interact with the presumed hydrophobic agonist binding site on the receptor. It is important that the more lipophilic local anesthetic bupivacaine (octanol-buffer partition coefficient 3420 [39]) inhibited lysophosphatidate signaling at significantly lower concentrations than did lidocaine (partition coefficient 366 [39]). (The agonist binding site might allow other anesthetic interactions, as we have shown recently that the lipophilic anesthetic propofol similarly interferes with lysophosphatidate signaling. [40]) Although the sigmoidal shape of the concentration-inhibition relation is compatible with a receptor site of action, the variability associated with high lysophosphatidate concentrations [7,14] makes it difficult to show competitive interactions conclusively. One other potential site of action deserves further study, namely a direct interaction with the lysophosphatidate molecule, or with lysophosphatidate binding to albumin, its physiologic carrier.

Our data indicate that commonly used local anesthetics can interfere significantly with lipid mediator signaling. Various lipid mediators (platelet-activating factor, prostaglandins, leukotrienes) are relevant in the perioperative context, making it important to understand their interactions with commonly used local anesthetics. This is of particular relevance to the newer, long-acting compounds.