Christian W. Honemann

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

UNLABELLED 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.

The Inhibitory Effect of Bupivacaine on Prostaglandin E 2 (EP 1 ) Receptor Functioning: Mechanism of Action

Prostaglandin E2 receptors, subtype EP1 (PGE2EP1) have been linked to several physiologic responses, such as fever, inflammation, and mechanical hyperalgesia. Local anesthetics modulate these responses, which may be due to direct interaction of local anesthetics with PGE2EP1 receptor signaling. We sought to characterize the local anesthetic effects on PGE2EP1 signaling and elucidate mechanisms of anesthetic action. In Xenopus laevis oocytes, recombinant expressed PGE2EP1 receptors were functional (half maximal effect concentration, 2.09 ± 0.98 × 10−6 M). Bupivacaine, after incubation for 10 min, inhibited concentration-dependent PGE2EP1 receptor functioning (half-maximal inhibitory effect concentration, 3.06 ± 1.26 × 10−6 M). Prolonged incubation in bupivacaine (24 h) inhibited PGE2-induced calcium-dependent chloride currrents (ICl(Ca)) even more. Intracellular pathways were not significantly inhibited after 10 min of incubation in bupivacaine. But ICl(Ca) activated by intracellular injection of GTP&ggr;S (a nonhydrolyzable guanosine triphosphate [GTP] analog that activates G proteins, irreversible because it cannot be dephosphorylated by the intrinsic GTPase activity of the &agr; subunit of the G protein) was reduced after 24 h of incubation in bupivacaine, indicating a G protein-dependent effect. However, inositol 1,4,5-trisphosphate- and CaCl2- induced ICl(Ca) were unaffected by bupivacaine at any time points tested. Therefore, bupivacaine’s effect is at phospholipase C or at the G protein or the PGE2EP1 receptor. All inhibitory effects were reversible. We conclude that bupivacaine inhibited PGE2EP1 receptor signaling at clinically relevant concentrations. These effects could, at least in part, explain how local anesthetics affect physiologic responses such as fever, inflammation, and hyperalgesia during the perioperative period.

Local Anesthetic Actions on Thromboxane-induced Platelet Aggregation

Some local anesthetics (LA), in concentrations present in blood during IV or epidural infusion, inhibit thrombus formation in the postoperative period. Studies on thromboxane A2 (TXA2) signaling in a recombinant model suggest that interference with TXA2-induced platelet aggregation may explain, in part, the antithrombotic actions of epidural analgesia and IV LA infusion. In this study we investigated the effects of clinically used LAs (lidocaine, ropivacaine, and bupivacaine) on TXA2-induced early platelet aggregation (1–5 s) by using quenched-flow and optical aggregometry. Our findings demonstrate that the LAs tested seem to have only a limited ability to inhibit TXA2-induced platelet aggregation assessed at early times (1–5 s). Therefore, the clinical effects of LAs on thrombi formation are unlikely to be explained by this manner alone. At large LA concentrations, moderate effects were obtained. Prolonged incubation with LA did not significantly increase effectiveness, and the lack of an effect could not be explained by generation of secondary mediators. The results were independent of the anesthetic studied. Local anesthetic effects on TXA2-induced early platelet aggregation (1–5 s) are unlikely to play a major role in the clinically observed antithrombotic effects of local anesthetics.

Volatile and Local Anesthetics Interfere with Thromboxane A2 Receptors Recombinantly Expressed in Xenopus Oocytes

It has been hypothesized that anesthetics induce malfunctions in some critical membrane proteins. New cell physiology research findings points to direct interactions with proteins. This implies a specific and direct mode of action.

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

ThromboxaneA2 (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 × 10−3 M), ropivacaine (IC50: ropivacaine 6.3 ± 0.9 × 10−5 M), or bupivacaine (IC50: 1.42 ± 0.08 × 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 × 10−5 M, R(+) ropivacaine 4.9 ± 1.7 × 10−4 M, S(−) ropivacaine 5.3 ± 0.9 × 10−5 M, and lidocaine 6.4 ± 2.8 × 10−4 M. Intracellular pathways activated by IP3 and GTPγS 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 × 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 × 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.

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 (ICl(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 ICl(Ca) if this current is regulated by phosphorylation. We therefore studied the effect of modulation of either PKC or protein phosphatases PP1&agr; and PP2A on ICl(Ca) stimulated either by lysophosphatidate (LPA) signaling or by microinjection of Ca. X. laevis oocytes were studied under voltage clamp. Rat PP1&agr; and PP2A were overexpressed in oocytes. PP, inositoltrisphosphate (IP3), the PP inhibitor okadaic acid (OA), the PKC inhibitor chelerythrine, or CaCl2 were directly injected into the oocyte. Responses to agonists (LPA 10−6 M, IP3 10−4 M, CaCl2 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 &agr;and PP2A inhibited ICl(Ca) from 7.6 ± 0.9 μC to 2.5 ±0.9 μC and 3.2 ±1.4 μC, respectively. PP inhibition enhanced ICl(Ca) in control oocytes and reversed the inhibitory effect in oocytes expressing PP1 &agr; or PP2A. PKC inhibition by chelerythrine enhanced both LPA-and CaCl2-induced ICl(Ca). Our data indicate that the Xenopus ICl(Ca) is modulated by phosphorylation. This may complicate design and interpretation of studies of G protein-coupled receptors using this model.