Carrie K. Chan

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.

Volatile anesthetic inhibition of neuronal Ca channel currents expressed in Xenopus oocytes

The genes encoding the alpha(1A), alpha(1B), alpha(1C) and alpha(1E) subunits of neuronal high voltage-gated Ca channels (HVGCCs) were separately expressed with beta(1B) and alpha(2)/delta subunits in Xenopus oocytes to determine the effects of volatile anesthetics (VAs) on currents through each specific channel. VA effects were determined on currents carried by Ba(2+) (I(Ba)) using the two electrode voltage clamp technique. Although time to peak was unaffected, both halothane (0.59 mM) and isoflurane (0.70 mM) reversibly inhibited peak I(Ba) by 25-35% and late current (at 830 ms) by 50-60%. A hyperpolarizing shift in steady-state inactivation of alpha(1E)-current was found which could contribute up to one third of observed decrease in the peak current. The rate of inactivation of I(Ba) seen with alpha(1A), alpha(1B) and alpha(1E)-type Ca channels was consistently increased by halothane and isoflurane. To more clearly quantify these effects, I(Ba) inactivation was fit by a single exponential function. The anesthetics depressed both the inactivating and non-inactivating residual components of I(Ba) and decreased the time constant of inactivation. In the case of I(Ba) through alpha(1C)-type channels, inactivation was minimal; however, the average current was inhibited by VAs. Similar inhibition of all these HVGCCs by halothane and isoflurane suggests that a common structural component may be involved. Furthermore, the inhibition of such neuronal HVGCCs in situ could alter synaptic neurotransmitter release and contribute to the anesthetic state.

Differential Inhibition of Lysophosphatidate Signaling by Volatile Anesthetics

Background Volatile anesthetics have been found to interfere with the functioning of several G protein‐coupled receptors, effects that may be relevant to the mechanism of anesthetic action. Lysophosphatidate (1‐acyl‐2‐sn‐glycero‐3‐phosphate; LP) is the simplest natural phospholipid. It has pronounced biological effects and signals through a specific G protein‐coupled receptor. Because of its lipophilicity, the LP receptor is a feasible site of anesthetic interaction. Therefore, the authors investigated the effects of halothane and isoflurane on LP signaling using Xenopus oocytes. Methods Mature oocytes were harvested from Xenopus frogs, isolated, and defolliculated manually. Lysophosphatidate receptors are endogenously present in these cells. Angiotensin receptors were expressed recombinantly to study anesthetic effects on intracellular signaling. Oocytes were studied individually with a two‐electrode voltage clamp at room temperature. Integrated Ca2+ ‐activated Cl sup ‐ currents (ICl(Ca)) were used to evaluate the effects of anesthetics on changes in intracellular Ca2+ concentration in response to receptor agonists (10 sup ‐7 M LP or 10 sup ‐7 M angiotensin II) or intracellular inositoltrisphosphate (IP3) injection. Results Halothane depressed LP signaling in a concentration‐dependent manner, with half‐maximal inhibition at 0.23 mM and virtually complete inhibition at 0.34 mM. Responses could be recovered after an anesthetic‐free wash. Oocyte injection with heparin, an IP3 receptor antagonist, completely blocked LP and angiotensin signaling, indicating similar IP3 ‐dependent pathways. However, ICl(Ca) induced by angiotensin receptor activation or intracellular IP3 injection were not inhibited by halothane. Isoflurane, at comparable concentrations, did not depress LP responses in oocytes significantly. Conclusions Lipid‐mediator signaling can be affected profoundly by volatile anesthetics. At clinically relevant concentrations, halothane and isoflurane have different effects on LP signaling. The inhibitory effects of halothane on the LP signaling pathway occur before the IP3 receptor.

Interactions between propofol and lipid mediator receptors : Inhibition of lysophosphatidate signaling

As a highly lipophilic drug, propofol may interact with lipophilic domains in addition to its likely primary site of action on the gamma-aminobutyrateA (GABAA) receptor. likely candidates for such interaction are the G protein-coupled membrane receptors for lipid intercellular mediators. The phospholipid lysophosphatidate (LP) has attracted attention as such a signaling molecule. It has a variety of biological actions, including vasoconstriction. We therefore studied the interaction between propofol and the LP receptor. Intracellular Ca2+ release in response to LP was assessed by measuring Cl- flux through Ca2+ -activated Cl- channels in Xenopus oocytes. The average charge movement in response to LP 10-7 M was 2.0 +/- 0.2 microCoulombs. Propofol in Intralipid[R] (0.01%) dose-dependently inhibited LP signaling (50% inhibitory concentration [IC50] 5.38 micro M). Propofol 28 micro M inhibited LP signaling by 81%. Intralipid[R] (0.01%) was without effect. To ascertain that intracellular signaling pathways and the Ca2+ -activated Cl (-) channel were not affected by propofol, we tested the effects of propofol (5.6 micro M) on currents induced by methylcholine (10-7 M) in oocytes expressing the m1 muscarinic acetylcholine receptor. No inhibition was observed. As both receptors share the same intracellular signaling pathway, we conclude that clinically relevant concentrations of propofol most likely inhibit the LP receptor or its G protein. Inhibition of LP signaling may explain some of propofols vasodilating actions. (Anesth Analg 1996;83:1090-6)