Gregor W. Nietgen

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. Conclusions: Lysophosphatidate signaling is inhibited by the extracellular application of lidocaine or bupivacaine. In contrast, inositoltrisphosphate or angiotensin signaling was not affected by local anesthetics. Therefore local anesthetics have a specific, extracellular effect on lysophosphatidate receptor functioning. As the local anesthetic concentrations used were similar to those observed after injection around surgical wounds, LP inhibition may play a role in the observed detrimental effects of local anesthetics on wound healing.

Intercellular Signaling by Lysophosphatidate Recent Developments

Lysophosphatidate (LPA) is an intercellular phospholipid messenger with a wide range of biologic effects. The first discovered source of LPA in the human body were activated platelets, but several other sites of LPA generation are now known. The number of cellular interactions is also growing steadily and responses to the compound range wide, from induction of mitogenesis to neurite retraction. LPA acts via a specific G protein-coupled receptor, of which one or more subtypes may exist. Intracellularly, this receptor activates several heterotrimeric G proteins. LPA induces cell proliferation via the small GTP-binding proteins ras, and triggers actin-based cytoskeletal events through rho. This review describes the most relevant recent developments in our understanding of LPA signaling.

synergistic Inhibition of Muscarinic Signaling by Ketamine Stereoisomers and the Preservative Benzethonium Chloride

Background: Ketamine (Ketalar; Parke‐Davis, Morris Plains, NJ) has been shown to inhibit muscarinic signaling with a median inhibitory concentration (IC50) of 5.7 micro Meter. Whereas Ketalar is a racemic mixture, recent interest has focused on clinical use of the S(+) ketamine isomer, which is three times as potent an analgesic as the R(‐) isomer yet seems to be associated with fewer psychoactive side effects. Therefore, the authors studied the effects of S(+) and R(‐) ketamine and the preservative benzethonium chloride on muscarinic signaling. Methods: Rat m1 muscarinic acetylcholine receptors were expressed recombinantly in Xenopus laevis oocytes. Ca2+ ‐activated Cl sup ‐ currents in response to 10 sup ‐7 M acetyl‐beta‐methylcholine were determined by two‐electrode voltage clamping in the presence of various concentrations of ketamine and benzethonium. Concentration‐inhibition curves were constructed and used for algebraic and isobolographic analysis. Results: The IC50 was 125 +/‐ 33 micro Meter for S(+) ketamine, and 91 +/‐ 19 micro Meter for R(‐) ketamine. This difference was not statistically significant, indicating that muscarinic inhibition by ketamine is not stereoselective. The R(‐)/S(+) mixture had an IC50 of 48 +/‐ 1 micro Meter, and thus the stereoisomers interact synergistically. When appropriate concentrations of benzethonium were added, an IC50 of 15 +/‐ 2 micro Meter resulted. Conclusions: The muscarinic inhibitory action of ketamine isomers is not stereoselective. Because S(+) ketamine is a significantly more potent analgesic, it should have less muscarinic inhibitory action than R(‐) ketamine when used in clinically equivalent doses. A significant fraction of the muscarinic inhibitory action of Ketalar is due to the preservative benzethonium. If reconstituted with a different preservative, Ketalar might be a less potent muscarinic antagonist.

Influence of anesthetics on endogenous and recombinantly expressed G protein-coupled receptors in the Xenopus oocyte

1. The oocyte of the African clawed toad (Xenopus laevis) offers a reliable, sensitive and disease resistant system to investigate recombinantly and endogenously expressed Ca2+ signaling G protein-coupled receptors and their intracellular signaling pathways. 2. To study receptor induced Ca2+ release, two-electrode voltage clamping can quantify a Ca2+-activated transmembrane Cl- current. Intracellular steps of the signaling pathway can be inhibited by injections of EDTA or heparin into the oocyte. Components of the intracellular pathway can be activated directly by GTPgammaS or IP3 injection. 3. We have investigated the effects of volatile, local and i.v. anesthetics on the signaling properties of the endogenous lysophosphatidate receptor and on mammalian receptors expressed recombinantly by intracellular injection of the encoding mRNA or cDNA. A number of receptors are sensitive to these anesthetics. Anesthetics interact with muscarinic, thromboxane A2 and lysophosphatidate signaling. 4. Investigations of the intracellular pathways revealed that the receptor or the receptor-G protein coupling is affected primarily and that mechanisms further downstream are not influenced by the various types of anesthetics.