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Anesth Analg 2002;95:88-97
© 2002 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

The Inhibitory Effect of Local Anesthetics on Bradykinin-Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells

Jinen Chen, MD*, Shuji Dohi, MD, PhD*, Zhiming Tan, MD, PhD*, Yoshiko Banno, PhD{dagger}, and Yoshinori Nozawa, MD, PhD{ddagger}

*Departments of Anesthesiology and Critical Care Medicine and {dagger}Biochemistry, Gifu University School of Medicine; and {ddagger}Gifu International Institute of Biotechnology, Gifu City, Gifu, Japan

Address correspondence and reprint requests to Shuji Dohi, MD, PhD, Department of Anesthesiology and Critical Care Medicine, Gifu University School of Medicine, Tsukasamachi-40, Gifu City, Gifu 500-8705, Japan. Address e-mail to shu-dohi{at}cc.gifu-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bradykinin induces activation of phospholipase D (PLD) via B2 receptors in neuronal cells. To demonstrate molecular mechanism(s) of local anesthetics, we examined whether and how local anesthetics affect bradykinin-induced PLD activation in PC12 cells. Using [3H]Palmitic acid-labeled PC12 cells stimulated with bradykinin, formation of [3H]phosphatidylbutanol was measured as a variable of PLD activity. Bradykinin-stimulated PLD activity seemed to peak at 2 min. Procaine, lidocaine, ropivacaine, bupivacaine, and tetracaine suppressed the bradykinin-induced PLD activation. We chose tetracaine, the most potent drug among the local anesthetics tested, to examine how local anesthetics affect phospholipase C, protein tyrosine kinase, and extracellular signal-regulated kinase, which are the molecules upstream of PLD. Tetra- caine at clinically relevant concentrations (1~10 x 10-4 M) inhibited the bradykinin-induced PLD activation in a dose- and time-dependent manner, but neither tetrodotoxin nor nifedipine affected the PLD activation. Tetracaine (5 x 10-4 M) slightly potentiated brady-kinin-induced phospholipase C activation. Bradykinin-stimulated protein tyrosine-phosphorylation and extracellular signal-regulated kinase activation were not affected by tetracaine. Tetracaine significantly decreased PLD activity of membrane fraction in PC12 cells. These results indicate that local anesthetics depress bradykinin-induced lipid signaling pathway(s) and may provide some clues to understanding the molecular mechanisms of these drugs for anesthesia or analgesia.

IMPLICATIONS: Local anesthetics depressed the bradykinin-induced activation of phospholipase D (PLD) in PC12 cells. The effects of tetracaine, the most potent among the anesthetics tested, on the bradykinin-induced intracellular signaling molecules were examined. The bradykinin-induced PLD activation could be one of the potential intracellular signaling molecular sites of local anesthetic action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bradykinin, a potent algogenic substance, induces pain by directly stimulating B2 receptors in skin, joint, and muscle afferents, as well as by sensitizing them to other stimuli (1). In the excitation of afferent fibers by bradykinin, protein kinase C (PKC) is thought to play a key role (2) and be involved in the activation of sodium channels (3) and calcium influx into sensory neurons (4). As a PKC activator, diacylglycerol (DG) is generated from activation of phospholipase C (PLC), which is also important in bradykinin-induced activation of sensory neurons (1) by acting on phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5,-trisphosphate (IP3) and DG (5). In addition, another source of DG is derived from activation of phospholipase D (PLD). PLD, an effective enzyme in the membrane lipid-mediated signal-transduction system, hydrolyzes phosphatidylcholine (PC), a major component of membrane phospholipids, to generate phosphatidic acid (PA) and choline. PLD activation also leads to the accumulation of DG via PA phosphohydrolase (6). The sustained DG formation is thought to induce a prolonged activation of PKC (6). Complimentary DNA cloning studies have revealed the existence of at least two PLD isozymes (1a, 1b, and 2) in mammalian cells (7). A putative PLD (oleic acid-dependent PLD), which is greatly activated by unsaturated fatty acids such as oleic acid, has also been found in PC12 cells (8).

Local analgesia achieved by injecting a local anesthetic into tissues, or in the proximity to the peripheral and central nervous systems, has been used to block pain transmission for nearly a century. Although it is generally accepted that local anesthetics exert their effects via blocking of voltage-gated Na+ channels and blocking of nociceptive impulses in the peripheral nerve fibers, other mechanisms are likely to be involved in their interruption of nociceptive conduction in the spinal cord (9). In addition to regional effects, local anesthetics are also infused IV for analgesia (9,10), but the underlying mechanisms cannot be explained merely by blockade of Na+ channels. Other molecular mechanisms may be involved in the actions of local anesthetics, for example, intracellular signal transduction molecules.

Our previous studies have demonstrated that local anesthetics interrupt the signal transduction via muscarinic receptors in PC12 cells (11) and that they can inhibit fMLP-induced PLD activation in neutrophil-like HL60 cells (12). In the present study, we observed that local anesthetics inhibited bradykinin-induced PLD activation. Through examining effects of tetracaine on the intracellular bradykinin signaling transduction, the bradykinin-induced PLD activation could be considered to be one of potential intracellular lipid signaling molecular sites for local anesthetic action.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Procaine hydrochloride, tetracaine hydrochloride, lidocaine hydrochloride, bupivacaine hydrochloride, nifedipine, tetrodotoxin (TTX), and bradykinin were purchased from the Sigma Chemical Company (St Louis, MO). Ropivacaine hydrochloride was obtained from Astra, Sodertalje, Sweden. Dulbecco’s modified Eagle’s medium and horse serum were obtained from Life Technologies (Grand Island, NY). Fetal bovine serum was obtained from Nippon Bio-supply Center (Tokyo, Japan). [9,10-3H]Palmitic acid (54.0 Ci/mmol), goat anti-mouse immunoglobulin G horseradish peroxidase-coupled secondary antibody, and enhanced chemiluminescence system used for Western blot analysis were obtained from Amersham Life Science (Buckinghamshire, UK). Antiphosphotyrosine mouse monoclonal antibody was obtained from Upstate Bio-technology Incorporated (Lake Placid, NY). [3H]Oleic acid, [palmitoyl-[3H]]dipalmitoyl PC and myo-[3H]inositol were obtained from DuPont New England Nuclear (Boston, MA). Antiphosphorylated-extracellular signal-regulated kinase (ERK) antibody was from New England Biolabs (Beverly, MA).

A PC12 cell line was kindly supplied by Dr Y. Sugimoto (Shirakawa, Institute of Animal Genetics, Fukushima, Japan). Monolayer cultures of the cells were maintained in 100-mm-diameter tissue culture dishes in Dulbecco’s modified Eagle’s medium supplemented with 10% (vol/vol) fetal bovine serum and 5% (vol/vol) horse serum in a humidified atmosphere containing 5% CO2 at 37°C. Stock cultures were subcultured routinely at a cell density of 2–3 x 106 cells/dish at least once a week, and culture media were renewed every 2 days.

PC12 cells were subcultured in 60-mm-diameter tissue culture dishes at 1 x 106 cells/dish and grown to confluency. The cells labeled with [3H]palmitic acid (2 µCi/dish) for 24 h were washed twice with buffer A (25 mM of HEPES, pH value of 7.4, 125 mM of NaCl, 5 mM of KCl, 1.2 mM of MgSO4, 2 mM of CaCl2, 1.2 mM of KH2PO4, 5 mM of glucose, and 1 mg/mL of bovine serum albumin) and preincubated in buffer A with or without the various reagents for the indicated duration at 37°C in the presence of 0.3% butanol (vol/vol). These cells were then stimulated with bradykinin. The reaction was terminated by aspiration of the buffer followed by the immediate addition of ice-cold phosphate-buffered saline (8% NaCl, 0.2% KCl, 2.88% Na2HPO4 · 12H2O, and 0.2% KH2PO4, at a pH value of 7.4)/methanol (2:5, vol/vol), and cells were scraped off the dishes with a scrapper, rubber policeman. Lipids were extracted according to the method of Bligh and Dyer and separated on Silica Gel LK6D thin-layer chromatography plates in a solvent system of the upper phase of ethyl acetate · 2,2,4-trimethylpentane · acetic acid · water (13:2:3:10, vol/vol), as described previously (13). The plates were exposed to iodine vapor, and [3H]phosphatidylbutanol (PBut), the variable of PLD activity, was identified by co-immigration with the PBut standard. The spots scraped off the plates were mixed with a scintillation mixture, and the radioactivity was counted in a liquid scintillation counter (Beckman LS-6500, Beckman Coulter, Inc).

Cells were scraped and washed with HEPES buffer (20 mM of HEPES, a pH value of 7.4, 5 mM of MgCl2, 1 mM of EGTA, 5 mM of dithiothreitol, 0.5 mM of phenylmethylsulphonyl fluoride, and 10 µg/mL of leupeptin) and then disrupted by N2 cavitation (600 psi at 4°C for 30 min). Unbroken cells and nuclei were then removed by centrifugation at 900g for 5 min, and the resulting supernatant was further centrifuged at 100,000g for 30 min to obtain pelleted membrane fractions. The membrane fractions were resuspended in HEPES buffer containing 100 mM of KCl and 0.5 mM of Mg/adenosine triphosphate and kept in a freezer (-80°C). PLD activity in the membrane fractions was measured by using the exogenous substrate phospholipid vesicles prepared as described previously (13). Mixed-lipid vesicles (phosphatidylethanolamine ·PIP2 · egg PC; 10:1.5:1, M ratio) containing [palmitoyl-[3H]] dipalmitoyl PC (3 µCi/mL) added to the membrane fractions in a reaction mixture containing 50 mM of HEPES (pH value of 7.5), 80 mM of KCl, 3 mM of MgCl2, 3 mM of EGTA, and 0.39 mM of CaCl2 and incubated with 10 µM of recombinant adenosine diphosphate-ribosylation factor and 100 µM of guanosine 5'-O-(3-thio-triphophate) (GTP{gamma}S) indicated concentrations of tetracaine in the presence of 0.3% butanol (vol/vol) at 37°C for 30 min. For the assay of oleate-dependent PLD activity, egg PC vesicles (20 µL) containing 15 nmol of egg PC and [palmitoyl-[3H]]dipalmitoyl PC (3 µCi/mL) were prepared by sonication and mixed with 5 µL of sodium oleate 12 mM. The substrate was added to the membrane suspension in a total volume of 120 µL containing 50 mM of HEPES (pH value of 7.0), 2 mM of EGTA, 500 mM of KCl, and 0.3% butanol (vol/vol) and then incubated at 37°C for 30 min. Reactions were terminated by the addition of 0.8 mL of chloroform/methanol (1:2, vol/vol), 0.4 mL of chloroform, and 0.3 mL of KCl 200 mM/EDTA 5 mM solution. The lipids were extracted, and the [3H]PBut formed was measured as described above.

Cells were labeled for 24 h with 2 µCi/mL of myo-[3H]inositol in an inositol-free medium. On the day of the experiment, cells were washed twice and preincubated in buffer A containing 10 mM of LiCl with or without local anesthetics for 30 min and then stimulated with 2 µM of bradykinin at 37°C. The reaction was terminated by the addition of 10% (vol/vol) perchloric acid. The cells were scraped off the dishes and removed to tubes. After neutralizing with 1.53 M of KOH/75 mM of HEPES, the tubes were centrifuged (900g for 5 min), and the aqueous phase was removed and diluted to 5 mL with H2O. The diluted extract was then applied to an AG1-X8 resin (formate) column. IP3 was eluted sequentially using 1.0 M of ammonium formate in 0.1 M of formic acid as eluent, and the radioactivity was counted in a liquid scintillation counter (Beckman LS-6500).

PC12 cells were subcultured in 60-mm-diameter tissue culture dishes at 1 x 106 cells/dish and grown for 4 days. The cells were washed twice with 2 mL of buffer A and preincubated in 3 mL of buffer A with indicated reagents at 37°C for 10 min. The cells were then stimulated with 2 µM of bradykinin at 37°C. The reaction was terminated by aspiration of the reaction buffer and washing twice with 2 mL of ice-cold phosphate-buffered saline. The washed cells were quickly scraped into 100 µL of buffer B (10 mM of Tris-HCl, a pH value of 7.4, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 150 mM of NaCl, 1 mM of EDTA, 0.5 mM of phenylmethylsulfonyl fluoride, 10 µg/mL of leupeptin, 1 mM of Na3VO4, 10 mM of NaF, and 0.1 mM of Na2MoO4) and transferred to a microcentrifuge tube. After incubation on ice for 30 min, the suspension was centrifuged at 13,000g for 20 min to obtain the cell extract. The proteins were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (10%) and electrophoretically transferred onto nitrocellulose membrane. After blocking the membranes with TBS-T (10 mM of Tris-HCl, a pH value of 7.5, 150 mM of NaCl, and 0.1% Tween 20) containing 5% bovine serum albumin, membranes were incubated with antiphosphotyrosine antibody or antiphosphorylated ERK antibody at room temperature for 90 min and then with the goat anti-rabbit immunoglobulin G horse-radish peroxidase-coupled secondary antibody at room temperature for 60 min. Detection was performed with enhanced chemiluminescence system.

Data were expressed as mean ± SD. Differences between values were analyzed using analysis of variance; when P < 0.05, differences were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bradykinin-Induced Activation of PLD in PC12 Cells
Stimulation of [3H]palmitic acid-labeled PC12 cells with bradykinin in the presence of butanol resulted in the generation of a PBut by transphosphatidylation of PLD. Upon bradykinin stimulation, PBut formation was increased in a dose-dependent manner with a maximum approximately 4.7-fold at 2 µM (Fig. 1A), and also time-dependently, reaching a plateau within 2 min (Fig. 1B).



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  		Figure 1. Bradykinin-induced phospholipase D (PLD) activation in PC12 cells. [3H]Palmitic acid-labeled cells were washed twice and then preincubated in buffer A in the presence of 0.3% butanol (vol/vol) for 10 min. (A) The cells were stimulated with indicated concentrations of bradykinin for 2 min at 37°C. *P < 0.01 versus 0 µM and **P < 0.001 versus 0 µM. (B) The PC12 cells were stimulated with 2 µM of bradykinin for indicated times. PLD activity was determined by measuring the formation of [3H]phosphatidylbutanol (PBut). Results are expressed as fold increase of control without bradykinin stimulation and given as mean ± SD of three different experiments performed in duplicate. *P < 0.01 versus 0 min and **P < 0.001 versus 0 min.

 
Inhibition of Bradykinin-Induced PLD Activity by Local Anesthetics
Procaine, lidocaine, ropivacaine, bupivacaine, and tetracaine at the same concentrations (5 x 10-4 M) all showed inhibitory effects on the bradykinin-induced PLD activation. Tetracaine had the greatest inhibitory effect on bradykinin-induced PLD activation (Fig. 2A). Thus, we chose tetracaine, the most potent anesthetic, in the following experiments. The bradykinin-induced PLD activation was inhibited by pretreatment with tetracaine in a dose- and time-dependent manner. Pretreatment with tetracaine at 1 x 10-4 M for 10 min showed some effect (P < 0.05) and at 5 x 10-4 M almost completely suppressed the activation of PLD (P < 0.0001) (Fig. 2, B and C).



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  		Figure 2. (A) Inhibitory effects of local anesthetics on the bradykinin-induced phospholipase D (PLD) activation in PC12 cells. [3H]Palmitic acid-labeled cells were pretreated with or without the 5 x 10-4 M of local anesthetics for 10 min and then stimulated with 2 µM of bradykinin for 2 min at 37°C in the presence of 0.3% butanol. *P < 0.05 versus control. PLD activity was determined by measuring the formation of [3H]phosphatidylbutanol (PBut). (B) and (C) The PC12 cells were pretreated with or without tetracaine at the indicated concentrations for the indicated times and then stimulated with 2 µM of bradykinin for 2 min at 37°C in the presence of 0.3% butanol. (A) The concentration dependency of tetracaine on bradykinin-induced PLD activation. *P < 0.05 versus 0 x 10-4 M, ***P < 0.0001 versus 0 x 10-4 M. (B) Time course of the inhibition of tetracaine on bradykinin-induced PLD activation. ***P < 0.0001 versus control. Data represent the mean ± SD of three different experiments performed in duplicate.

 
Effects of TTX and Nifedipine on Bradykinin-Induced PLD Activity
TTX, a blocker of Na+ channels, did not affect bradykinin-induced PLD activation in PC12 cells (Fig. 3). Depletion of extracellular Ca2+ almost completely prevented the bradykinin-stimulated activation of PLD (Fig. 3). However, nifedipine, a blocker of L-type Ca2+ channels, did not suppress the bradykinin-induced [3H]PBut formation (Fig. 3).



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  		Figure 3. Effect of tetrodotoxin (TTX), extracellular Ca2+, and nifedipine on the bradykinin-phospholipase D (PLD). PC12 cells were labeled with [3H]palmitic acid for 12 h and then washed twice with buffer A (Ca2+-free means added 2 mM of EGTA in buffer A). The cells were treated with or without 5 x 10-4 M of tetracaine, 10 µM of TTX, or 10 µM of nifedipine in the presence of 0.3% butanol (vol/vol) for 10 min and then stimulated with 2 µM of bradykinin for 2 min. The amount of [3H]phosphatidylbutanol (PBut) formed was quantified as described in Materials the Methods. Data are mean ± SD from three different experiments performed in duplicate determinations. ***P < 0.0001 versus control.

 
Effects of Tetracaine on Bradykinin-Induced PLC Activity
Bradykinin at 2 µM also stimulated generation of inositol monophosphate (IP1) and IP3 in PC12 cells. The IP1 formation increased in a time-dependent manner and the IP3 formation maximally observed at 1 min. Interestingly, both IP1 and IP3 formations by bradykinin were potentiated by the tetracaine pretreatment (Fig. 4, A and B).



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  		Figure 4. Effect of tetracaine on bradykinin-induced phospholipase C (PLC) activation in PC12 cells. myo-[3H]inositol-labeled PC12 cells were preincubated in buffer A with or without tetracaine at 5 x 10-4 M for 10 min and then stimulated with 2 µM of bradykinin for the indicated times at 37°C. (A) Inositol monophosphate (IP1) formation and (B) inositol triphosphate (IP3) formation were measured. Data represent the mean ± SD from three different experiments performed in duplicate. *P < 0.05 versus control and **P < 0.001 versus control.

 
Effects of Tetracaine on Bradykinin-Induced Protein Tyrosine-Phosphorylation and ERK Phosphorylation
In PC12 cells, genistein, an inhibitor of protein tyrosine kinase, almost completely prevented the bradykinin-induced PLD activation (Fig. 5A), suggesting that tyrosine kinase is involved in the PLD activation. Bradykinin stimulation of PC12 cells induced tyrosine phosphorylations of several proteins, but tetracaine (5 x 10-4 M) did not affect the bradykinin-induced protein tyrosine-phosphorylation (Fig. 5B). Stimulation of PC12 cells with 2 µM of bradykinin induced strong phosphorylation of ERK1 and ERK2. PD98059, an inhibitor of ERK pathway, inhibited the phosphorylation of ERKs (Fig. 6) and showed a partial inhibitory effect on the bradykinin-induced PLD activation (Fig. 6A), indicating that ERK is somehow involved in the PLD activation. Although 5 x 10-4 M of tetracaine decreased carbachol-stimulated phosphorylation of ERKs, as previously reported (Fig. 6C), tetracaine (5 x 10-4 M) did not show any inhibitory effect on the bradykinin-induced phosphorylation of ERKs (Fig. 6D). Genistein inhibited both bradykinin- and carbachol-induced phosphorylations of ERKs (Fig. 6, C and D). It is the suggested difference between the mechanisms of local anesthetics’ action on the carbachol- and bradykinin-stimulated signaling pathways.



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  		Figure 5. (A) Genistein inhibited bradykinin-induced phospholipase D (PLD) activity. Radio-labeled PC12 cells were pretreated with or without 100 µM of genistein for 10 min and stimulated by 2 µM of bradykinin for 2 min. [3H]phosphatidylbutanol (PBut) formation was determined. Data represent the mean ± SD from three different experiments performed in duplicate. ***P < 0.0001 versus control. (B) Effect of tetracaine on bradykinin-induced protein tyrosine-phosphorylation. PC12 cells were treated with or without 5 x 10-4 M of tetracaine for 10 min and then stimulated with or without 2 µM of bradykinin for 2 min. Extracted proteins (50 µg) were analyzed with Western Blot analysis with antiphosphotyrosine antibody. The arrows on the left indicate phosphorylated proteins.

 


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  		Figure 6. (A) PD98059 (PD) suppressed bradykinin-induced phospholipase D (PLD) activity. Radio-labeled PC12 cells were pretreated with PD or without (Control) 50 µM of PD98059 for 30 min and then stimulated with 2 µM of bradykinin for 2 min. PLD activity was examined. Data represent the mean ± SD from three different experiments performed in duplicate. **P < 0.001 versus control. (B) Effect of PD98059 on bradykinin-induced extracellular signal-regulated kinase (ERK) phosphorylation in PC12 cells. (C) and (D) Different effect of tetracaine on bradykinin- or carbachol-induced ERK phosphorylation in PC12 cells. The cells were treated with or without 5 x 10-4 M of tetracaine for 10 min and then stimulated with or without 1 mM of carbachol (C) or 2 µM of bradykinin (D) for 2 min. Extracted proteins (30 µg) were used in Western Blot analysis with antiphosphospecific-ERK antibody. The arrows on the left indicate phosphorylated ERK1 and ERK2.

 
Effect of Tetracaine on PLD Activation in Membrane Fraction from PC12 Cells
The PLD activity was examined in the membrane fractions isolated from PC12 cells in the presence or absence of GTP{gamma}S and PIP2 or oleate. The PIP2-dependent PLD activity was increased sevenfold and was not affected by GTP{gamma}S. Tetracaine 5 x 10-4 M significantly decreased the PIP2-dependent PLD activity with or without GTP{gamma}S (Fig. 7A). Oleic acid also stimulated PLD activity in the membrane fraction (Fig. 7B). Tetracaine dose-dependently inhibited the PIP2-dependent PLD activity with a maximum at 1 mM but did not show a significant effect on the oleic acid-dependent PLD activity in the membrane fractions (Fig. 7C).



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  		Figure 7. Effects of tetracaine on the phospholipase D (PLD) activity of the membrane fraction from PC12 cells. The membrane fractions were prepared and incubated with the substrate phospholipid vesicles as described in Materials and Methods at 37°C for 30 min in the presence of 0.3% butanol. (A) Inhibitory effect on guanosine 5'-O-(3-thio-triphophate) (GTP{gamma}S)-independent PLD. Tetracaine 5 x 10-4 M was or was not included in the reaction mixture and with or without 10 µM of GTP{gamma}S. *P < 0.01 versus the absence of tetracaine. (B) Oleic acid-dependent PLD activity. The PLD activity was performed in the presence or absence of 1.8 mM of sodium oleac acid (OA). **P < 0.001 versus the absence of sodium oleate. (C) Effect of tetracaine on the phosphatidylinositol 4,5-bisphosphate (PIP2)- and oleate-dependent PLD activities in the membrane fraction. The reaction mixtures were incubated with the indicated concentration of tetracaine in the presence of PIP2 or oleic acid in the substrate phospholipid vesicles. PLD activity was determined by measuring the formation of [3H]phosphatidylbutanol (PBut). Data represent the mean ± SD from three different experiments performed in duplicate. *P < 0.01 versus 0 x 10-4 M and **P < 0.001 versus 0 x 10-4 M.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding of this study is that tetracaine inhibited bradykinin-induced PLD, and that tetracaine’s action seemed to be independent with the suppression of bradykinin-induced PLC-PKC activation or ERK phosphorylation. Neurotransmitter- and neuromodulator-mediated activation of PLD in the brain and many types of neural-derived cells (14), and bradykinin-induced PLD activation, have been acknowledged as components of the receptor-mediated signal transduction in several cell lines (15,16).

Because local anesthetics block nerve conduction via acting at Na+ channels, we considered that they might prevent bradykinin-induced PLD activity by their blocking effect on Na+ channels. However, TTX, a potential blocker of Na+ channels, showed no effect on bradykinin-induced PLD activation in PC12 cells. Local anesthetics have also been reported to block L-type-specific Ca2+ channels (17), and bradykinin-mediated responses exhibited a requirement for extracellular Ca2+ influx (18). These findings raised the possibility that local anesthetics would block Ca2+ influx and then inhibit PLD activation. Although depletion of extracellular Ca2+ abrogated the activation of bradykinin-induced PLD, the action of local anesthetics on PLD was unlikely to be associated with its effect on L-type Ca2+ channels because the L-type Ca2+ channel blocker, nifedipine, did not affect bradykinin-induced [3H]PBut formation in PC12 cells.

Activation of PLC occurs via heterotrimetric Gq protein-coupled B2 receptors stimulated with bradykinin (1) and hydrolyzes PIP2 to produce IP3 and DG, each of which mobilizes intercellular calcium and activates the PKC pathway, respectively (19). Because PLD is regulated by PKC (20) and because local anesthetics have been suggested to inhibit directly or indirectly PLC activation (21,22), it was considered that local anesthetics inhibit PLD activity, at least partially, by their inhibitory effects on the bradykinin-induced PLC-PKC pathway. However, the results of the present study showed that bradykinin-stimulated PLC activity was not inhibited, but rather was potentiated, by 0.5 mM of tetracaine. Therefore, it seems likely that the inhibitory effect of tetracaine on the PLD activation is independent from the PLC pathway in PC12 cells.

We have reported that local anesthetics, including tetracaine, inhibited carbachol-induced protein tyrosine phosphorylation in PC12 cells (11). Several lines of evidence have been accumulated to indicate that tyrosine kinase would be involved in PLD activation in several cell systems (23,24). The activation of PLD by carbachol in PC12 cells is inhibited by tyrosine kinase inhibitors, including genistein, and indicates that tyrosine phosphorylation is associated with the stimulation of PLD (25). In the present study, bradykinin-stimulated PLD activation was completely inhibited by the tyrosine kinase inhibitor genistein. However, tetracaine did not affect bradykinin-induced protein tyrosine phosphorylation, suggesting that the inhibitory effect of local anesthetics on bradykinin-stimulated PLD would not be dependent on protein tyrosine phosphorylation. In addition, ERKs have been reported to be involved in H2O2-induced PLD activation in PC12 cells (25), and we have demonstrated, in the present study, that the bradykinin-induced PLD activity was partly inhibited by PD98059, which completely inhibited the bradykinin-induced ERK activation (Fig. 6). In our previous study, we indicated that local anesthetics prevent muscarinic receptor-mediated ERK activation and protein phosphorylation in PC12 cells (11). These observations lead us to assume that local anesthetics inhibited PLD activity by preventing ERK activation. However, because tetracaine did not affect bradykinin-induced ERK phosphorylation, the results would also suggest that the site(s) of tetracaine’s action should not be located upstream of ERKs.

Local anesthetics affect many membrane-associated proteins, such as Na+ channels, adenylate cyclase, guanylate cyclase, ion-pumping enzymes, phospholipase A2, and PLD1 (12,26). The actions of these drugs on membrane-associated proteins are thought to result from conformational changes of proteins via hydrophobic interaction and the interaction with the lipids surrounding them (26). It is conceivable that local anesthetics in clinically relevant concentrations would inhibit bradykinin-induced PLD2 activity by the same mechanism(s) to suppress the PLD2 activity in PC12 cell membrane.

Although two isoforms of PLD have been identified in mammalian cells (PLD1 and PLD2) and both require PIP2 for their in vitroactivation (6), PLD1 shows low basal activity and is activated by the small GTP-binding proteins, adenosine diphosphate-ribosylation factor, and Rho, whereas PLD2 is constitutively active and is not responsive to small G proteins (6,7). The isoform expressed in PC12 cells was reported to be only PLD2 (27), and we have examined PLD activity in the membrane fraction from PC12 cells in the present study. The PLD activation required PIP2 but was independent of GTP{gamma}S (Fig. 7A), suggesting that the membrane fraction PC12 cells contain PLD2 but not PLD1. That there was no effect of tetracaine on oleic acid-dependent PLD activation in PC12 membrane fraction may support that tetracaine produces an inhibitory effect on PLD2 in PC12 cells.

PLD plays physiological and pathophysiological roles in the nervous system (14). PLDs initiate the formation of lipid-derived intra- and intercellular mediators by hydrolysis of PC. The primary lipid product of PLD is PA, which exhibits a number of biological activities, including vesicular transport and cytoskeleton rearrangement (28). Further metabolism of PA by PA phosphohydrolase leads to the generation of DG for activation of PKC in the nervous system (29). Prolonged activation of PKC in the nervous system was considered to be associated with long-term potentiation (30). The other product, choline, from PLD activation is a precursor of the neurotransmitter acetylcholine (31). Bradykinin, as one of the most potent endogenous algesic substance-mediated hyperalgesia, involves the production of DG and PKC activation (32). Thus, DG generated from PLD activation should be further investigated to delineate the role of PLD activation in bradykinin-mediated physiological and pathophysiological processes Fig. 8.



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  		Figure 8. Illustration of bradykinin-induced phospholipase C (PLC) and D (PLD) cascade. Activation of PLC occurs via heterotrimetric Gq proteins-coupled B2 receptors stimulated with bradykinin (1), and hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol triphosphate (IP3) and diacylglycerol (DG), which regulates Na+ and Ca2+ channels. PLD hydrolyzes phosphatidylcholine (PC) to generate phosphatidic acid (PA). Further metabolism of PA by PA phosphohydrolase leads to generation of DG for prolonged activation of protein kinase C (PKC), which may be also involved with the regulating Na+ and Ca2+ channels. In the presence of butanol, PBut is produced instead of PA. Protein tyrosine kinase (PTK) and ERK are upstream of PLD.

 
Local anesthetics are widely used in anesthesia and analgesia. Aside from blockade of Na+ channels, non-Na+ channel mechanisms of local anesthetics’ action have also been reported, for example, to cause blockage of N-methyl-D-aspartate receptors and substance P receptors (9) and to suppress superoxide production from neutrophils (33). The usual concentrations of three major local anesthetics for peripheral and central anesthesia are 1%–5% lidocaine (approximately 37–185 mM), 0.5%–0.75% bupivacaine (approximately 15–23 mM), and 0.25%–0.5% tetracaine (approxi-mately 8–17 mM), respectively. Na+ currents in myelinated nerves in vitro were initially inhibited by lidocaine at 0.2 mM or bupivacaine at 0.025 mM and completely blocked by the drugs at clinical doses (34). The concentrations of local anesthetic-inhibited nerve conduction in vitro seem to be smaller than the one that inhibited bradykinin-induced PLD. Otherwise, lidocaine at 0.4 mM in vivo inhibited impulses of afferent fiber in the rat (34). The plasma concentration of lidocaine in systemic analgesia is thought to be <10 µg/mL (approximately 0.04 mM) (9). The mean concentration of tetracaine in human cerebrospinal fluid during spinal anesthesia is reported to be approximately 0.02–0.4 mM (35). Based on these observations, it is possible that the effect of local anesthetics on PLD could be involved with spinal anesthesia or analgesia.

The rank order of local anesthetics for suppression of PLD activation seems to agree with their action on the muscarinic receptor-mediated ERKs phosphorylation (11), PLD1 activation in HL60 cells (12), and superoxide production of neutrophils as well (33). We may speculate that potency of local anesthetics could pertain to non-Na+ channel-mediated action, such as action on intracellular signal molecules. Differences in the suppression of muscarinic receptor-mediated ERKs activation and B2 receptor-mediated ERKs activation could be explained, though not entirely clearly, by different mechanism(s) involved in the intracellular signaling pathways.

In the present study, local anesthetics inhibited the bradykinin-induced PLD activation, and tetracaine showed the most potent effect. TTX and nifedipine did not affect the bradykinin-induced PLD, which may suggest the action of local anesthetics was not by their blocking effect on Na+ or L-type Ca2+ channels. The effects of tetracaine on the bradykinin-induced PLC, protein tyrosine kinase, and ERK signaling molecules upstream of PLD were not observed. Otherwise, tetracaine showed an inhibitory effect on GTP{gamma}S-independent PLD activation, whereas it did not affect oleic acid-dependent PLD. The above results may suggest that local anesthetics do not depress the bradykinin-induced PLD2 by their effects on Na+ or L-type Ca2+ channels and upstream molecules in PC12 cells. The bradykinin-mediated PLD activation could be an intracellular lipid signaling molecular sites for local anesthetic action.


   Acknowledgments
 
Supported, in part, by research grants (Nos. 11307027 and 14207059) from the Ministry of Education, Science, and Culture of Japan, Tokyo, Japan.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Accepted for publication March 19, 2002.





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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press