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Anesth Analg 2003;96:1656-1660
© 2003 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

The Effects of Local and Intravenous Anesthetics on Recombinant Rat VR1 Vanilloid Receptors

Kazuyoshi Hirota, MD*, Darren Smart, PhD{dagger}, and David G. Lambert, PhD{ddagger}

*Department of Anesthesiology, University of Hirosaki School of Medicine, Japan; {dagger}Neurology-CEDD, GlaxoSmithKline, Essex; and {ddagger}University Department of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, United Kingdom

Address correspondence and reprint requests to David G. Lambert, PhD, University Department of Anesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE1 5WW, UK. Address e-mail to DGL3{at}le.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Capsaicin, acting at the vanilloid 1 receptor (VR1), may potentiate local anesthetic activity, and as a ligand-gated ion channel of the transient receptor potential family, may also be a target for IV general anesthetics. We have examined whether local (lidocaine, prilocaine, and procaine 0.1–10 mM; 10 mM represents 0.25%–0.27% wt/vol) or IV anesthetics (propofol 10 µM, thiopental 100 µM, and ketamine 100 µM) interact with recombinant rat VR1 expressed in human embryonic kidney (HEK293) cells (VR1-HEK293). We have assessed receptor interaction functionally by monitoring intracellular Ca2+ ([Ca2+]i) in Fura2-loaded cells at 37°C. The addition of capsaicin (60 nM) produced a time-dependent biphasic increase in [Ca2+]i amounting to 50–100 nM above than basal, which was inhibited by capsazepine 10 µM and was absent in wild type HEK293 cells. Lidocaine and prilocaine alone (e.g., at 10 mM) significantly increased [Ca2+]i by 67 ± 6 nM and 33 ± 7 nM, respectively, and concentration-dependently inhibited the capsaicin response. The effects of procaine were obscured by anesthetic-induced quenching of Fura2. In wild type HEK293 cells, lidocaine (10 mM) alone produced a small increase in [Ca2+]i. All IV anesthetics failed to modify capsaicin-increased [Ca2+]i. In conclusion, the present data suggest that local but not IV general anesthetics interact with recombinant rat VR1 receptors with the former anesthetics having antagonistic activity.

IMPLICATIONS: Vanilloid receptors (VR1) are activated by capsaicin, the pain-producing component of hot chili peppers. We suggest that local (but not IV general) anesthetics may have inhibitory actions on this receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Capsaicin, the pungent agent in chili peppers, activates vanilloid 1 receptors (VR1) to produce itching, pricking, and burning sensations. This ligand-gated ion channel, of the transient receptor potential (trp) family, is important in the nociceptive system because VR1 knockout mice (mice genetically manipulated to lack the VR1 receptor) demonstrate impaired nociception (1). Topical capsaicin is used in experimental hyperexcitability models (2). Capsaicin selectively stimulates, and then depletes, substance P release from nociceptive primary afferents (3) and inhibits tetrodotoxin-resistant Na+ currents, which are found primarily in small-diameter nociceptive afferents (4). Because these pharmacological properties may produce pain relief, topical capsaicin has been used clinically in the treatment of neuropathic pain, such as postherpetic neuralgia and diabetic neuropathy, although its efficacy (and effective dose) has been questioned (3).

Kohane et al. (5) reported that capsaicin may potentiate the in vivo local anesthetic activity of percutaneously injected site-1 Na+ channel blockers, suggesting that a combination of local anesthetics with capsaicin may produce improved pain relief. In addition, because VR1 is a member of the ligand-gated ion channel family of a class different (trp) to that to which the {gamma}-aminobutyric acid (GABA)A receptor belongs (6), it will be interesting to examine the effects of IV general anesthetics at VR1 receptors.

In the present study, we therefore examined whether local (lidocaine, prilocaine, and procaine) and IV anesthetics (propofol, thiopental, and ketamine) interact with recombinant rat VR1 expressed in human embryonic kidney (HEK293) cells (VR1-HEK293). We assessed receptor interaction in functional responses at the VR1 by monitoring intracellular Ca2+ in Fura2-loaded cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
VR1-HEK293 cells (7) were maintained in minimum essential medium supplemented with 0.2 mM of glutamine, 50 IU/mL of penicillin, 50 µg/mL of streptomycin, 2.5 µg/mL of Fungizone, and 10% fetal calf serum. Cultures were maintained at 37°C in 5% CO2/humidified air, fed every 2–3 days, passaged every 5–6 days, and used up to passage 24.

Intracellular Ca2+ ([Ca2+]i) was measured as previously described (8). Confluent VR1-HEK293 cells were detached with harvest buffer (EDTA 1.7 mM, NaCl 154 mM, and HEPES 10 mM) then washed twice with and resuspended into Krebs-HEPES buffer (NaCl 143.4 mM, KCl 4.8 mM, HEPES 10 mM, CaCl2.2H2O 2.6 mM, KH2PO4 1.2 mM, MgSO4.7H2O 1.2 mM, and glucose 11.7 mM). Suspensions of cells were then incubated in Krebs-HEPES buffer containing Fura2-AM (5 µM) for 30 min at 37°C, followed by 20 min postincubation at room temperature to allow for complete de-esterification. Loaded cells were then resuspended in Krebs-HEPES buffer supplemented with 0.2% bovine serum albumin and maintained on ice. Cuvette temperature was adjusted to 37°C using a circulating water bath whose temperature was monitored by thermocouple thermometer. Finally, [Ca2+]i was fluorometrically measured in a PerkinElmer LS 50B fluorometer (PerkinElmer, Beaconsfield, UK) with excitation wavelengths set at 340 and 380 nm and emission set at 510 nm. [Ca2+]i was calculated from the 340:380 ratio, according to Grynkiewicz et al. (9), where Rmax and Rmin were determined using Triton x 100 (0.1% vol/vol) and EGTA (4.5 mM; pH value > 8.0).

Cell suspensions (total volume, 2 mL) were allowed to equilibrate for 3 min before the administration of local anesthetics (0.1–10 mM). A 10-mM concentration roughly corresponds to 0.25%–0.27% wt/vol (Table 1). One minute later, capsaicin (60 nM) was added. Capsaicin 60 nM was chosen from our previous study (8) and represents a marginally sub-50% effective concentration (EC50) (126 nM) at this temperature. In some experiments, the VR1 antagonist capsazepine (10 µM) was included at least 5 min before capsaicin addition. In addition, we also examined the effects of capsaicin and lidocaine in wild type HEK293 cells. To determine whether capsaicin- or lidocaine-increased [Ca2+]i was due to Ca2+ influx, changes in [Ca2+]i in cells suspended in Ca2+ free Krebs-HEPES buffer with 0.5 mM of EGTA added was monitored. In some experiments, CaCl2 2 mM was re-added after capsaicin- lidocaine.


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  		Table 1. Effects of Prilocaine and Procaine on Basal- and Capsaicin- (60 nM) Induced Increase in [Ca2+]i HEK293-VR1 Cells
 
One minute before the addition of 60 nM of capsaicin, thiopental (100 µM), propofol (10 µM), ketamine (100 µM), and a nonanesthetic barbiturate barbituric acid (2 mM) were added. These concentrations were chosen because they represent the maximum (with the exception of barbituric acid used as a negative control barbiturate) that would be encountered clinically.

Data are presented as either a typical trace or as mean ± SEM (from individual experiments). [Ca2+]i is presented either as raw total or as change, {Delta}Ca2+, after basal subtraction. Statistical analysis was by analysis of variance and Student’s t-test as appropriate with P < 0.05 considered as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In VR1-HEK293 cells, the addition of 60 nM of capsaicin produced a prompt time-dependent biphasic increase in [Ca2+]i. This response peaked at 15~20 s and amounted to some 50–100 nM above basal. Lidocaine (Fig. 1) and prilocaine (Table 1) per se significantly increased [Ca2+]i. The increase was greater with lidocaine (e.g., 10 mM of lidocaine and procaine increased [Ca2+]i 67 ± 6 nM and 33 ± 7 nM, respectively; P < 0.05). Both lidocaine (Fig. 1) and prilocaine (Table 1) significantly inhibited capsaicin-increased [Ca2+]i in a concentration-dependent manner. Moreover, this inhibition at the largest concentration (10 mM) was complete. Because procaine quenched Fura2 fluorescence, the effects of procaine at 10 mM could not be accurately assessed. However, at 1 mM, the quenching effect was small, revealing an inhibition of the capsaicin-increased [Ca2+]i response (Table 1).



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  		Figure 1. Effects of lidocaine (Lid) on basal and capsaicin-induced (60 nM) increase in intracellular Ca2+ ([Ca2+]i) in human embryonic kidney (HEK293)-vanilloid receptor (VR1) cells. In the upper panel, a typical trace is depicted, whereas in the lower panel, data are mean ± SEM (n = 5). **P < 0.05 increased compared with basal; *P < 0.01 reduced compared with capsaicin. If calculated based on the weight of the hydrochloride salt, lidocaine 10 mM, 1 mM, and 0.1 mM yields equivalent percentage concentrations of 0.271%, 0.0271%, and 0.00271%, respectively.

 
Capsazepine 10 µM completely inhibited capsaicin-increased [Ca2+]i but failed to affect the lidocaine-induced increase in [Ca2+]i (Fig. 2), indicating that the lidocaine increase in [Ca2+]i was independent of the VR1 receptor. To test this hypothesis, we examined the effects of capsaicin (60 nM as a control) and lidocaine (10 mM) in wild type (nontransfected) HEK293 cells. In these cells, lidocaine but not capsaicin increased [Ca2+]i (data not shown), confirming that lidocaine was not acting at the VR1 receptor to directly increase [Ca2+]i.



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  		Figure 2. Capsaicin- (60 nM) but not lidocaine-induced (Lid, 10 mM) increase in [Ca2+]i is antagonized by capsazepine (CPZ, 10 µM) in human embryonic kidney (HEK293)-vanilloid receptor (VR1) cells. In panels A and B, typical traces are depicted, and in panel C, mean ± SEM (n = 4) are shown. *P < 0.05 reduced compared with capsaicin.

 
If the lidocaine-induced increase in [Ca2+]i is not caused by an interaction with the VR1 receptor, is the increase in [Ca2+]i from intracellular or extracellular sources? To test this, we repeated the capsaicin (60 nM) and lidocaine (10 mM) responses in cells resuspended in Ca2+-free buffer supplemented with 0.5 mM of EGTA. This treatment would be expected to produce extracellular-free Ca2+ concentrations in the low nanomolar range. Under these conditions, the capsaicin response was abolished and the lidocaine response reduced by some 84% (Fig. 3). Note also that there was a readjustment of basal [Ca2+]i in Ca2+ free buffer. The addition of CaCl2 produced a prompt and substantial increase in [Ca2+]i (Fig. 3). Moreover, there was a clear overshoot of the [Ca2+]i response for both lidocaine and capsaicin.



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  		Figure 3. Both capsaicin (60 nM) and lidocaine (Lid, 10 mM) stimulate Ca2+ influx in human embryonic kidney (HEK293)-vanilloid receptor (VR1) cells. In the absence of added extracellular Ca2+, the responses to capsaicin and lidocaine are lost but can be restored by re-addition of 2 mM of CaCl2. In the upper panel, a typical trace is depicted, whereas in the lower panel, data are mean ± SEM (n = 4). P < 0.05 reduced compared with capsaicin or lidocaine.

 
Preincubation of VR1-HEK293 cells with thiopental (100 µM), propofol (10 µM), ketamine (100 µM), or barbituric acid (2 mM) did not significantly affect [Ca2+]i (data not shown). In addition, the response to 60 nM of capsaicin was similarly unaffected (Table 2).


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  		Table 2. Effects of IV Anesthetics on Capsaicin- (60 nM) Induced Increase in [Ca2+]i HEK293-VR1 Cells
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have clearly demonstrated that local anesthetics (lidocaine, prilocaine, and procaine) concentration-dependently inhibit capsaicin-induced increases in [Ca2+]i in VR1-HEK293 cells. No such effect was observed for a range of IV general anesthetics (thiopental, propofol, and ketamine). In addition and not of any obvious clinical relevance, there is the interesting direct stimulatory effect of lidocaine and prilocaine where lidocaine at least gates extracellular Ca2+ per se.

Kohane et al. (5) reported that co-administration of tetrodotoxin site 1 Na+ channel blockers and capsaicin produce synergistic effects on nerve block in vivo. In addition, they demonstrated that this synergism may be caused by different inhibitory effects of tetrodotoxin and capsaicin on Na+ currents mediated by tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ channels, respectively. These authors also concluded that the synergistic interactions may be useful in developing prolonged local anesthetics and elucidating mechanisms of functionally selective nerve block. Shin et al. (10) also showed that capsaicin could potentiate impulse block by lidocaine in the isolated sciatic nerve. However, as the present study suggests, although local anesthetics may inhibit activation of VR1 receptors by capsaicin, it is unlikely that co-administration of local anesthetics and capsaicin would produce synergistic effects. Thus, we speculate that this inhibitory interaction of local anesthetic with VR1 receptors may be the reason why Kohane et al. (5) failed to show a strong synergistic interaction between capsaicin and bupivacaine. However, it should also be noted that in the present study, we have used VR1 receptors heterologously expressed in HEK293 cells. Therefore, it seems from this study that local anesthetics (at least lidocaine, prilocaine, and, to a certain extent, procaine) act to inhibit VR1 responses. This is not surprising, because local anesthetics are known to inhibit voltage-gated Na+ channels in a use-dependent manner (11), and thus, we could envisage the local anesthetic molecule occupying the channel either directly or indirectly, although we have no experimental evidence to support this possibility. Additional studies to examine whether the effects of lidocaine on the capsaicin response are competitive would be interesting and may yield information as to the site of action (intracellular versus extracellular) as capsaicin binds to the intracellular portion of the receptor (12).

The increase in [Ca2+]i in VR1-HEK293 cells by lidocaine was markedly reduced by removal of extracellular Ca2+. However, a small but significant increase in [Ca2+]i was still observed. Thus, extracellular Ca2+ may play an important role in the increase in [Ca2+]i, although lidocaine may induce Ca2+ release from [Ca2+]i stores. Gold et al. (13) reported that lidocaine induced an increase in the [Ca2+]i in isolated rat dorsal root ganglion neurons (EC50 = 21 mM) via Ca2+ influx across the plasma membrane and Ca2+ release from intracellular stores. In contrast, in the present study, removal of extracellular Ca2+ completely inhibited capsaicin-increased [Ca2+]i. As nonselective cation channels, VR1 receptors are known to be activated by noxious heat, extracellular protons, and vanilloid compounds, and their activity are absent in Ca2+-free conditions (14).

Franks and Lieb (6) demonstrated that ligand-gated ion channels may be sensitive to clinically relevant concentrations of general anesthetics with effects reported at GABAA, glycine, and nicotinic acetylcholine receptors. In the present study, clinically relevant concentrations of a range of IV anesthetics did not affect the capsaicin-induced increase in [Ca2+]i in VR1-HEK293 cells. As a member of the trp family of receptors, it is clear from the present study that the VR1 receptor is insensitive to the IV anesthetics used in this study. However, Tsutsumi et al. (15) reported that propofol activated VR1 receptors in VR1-HEK293 cells. Although they showed that propofol per se increased [Ca2+]i in VR1-HEK293 cells, the EC50 was 170 ± 27 µM, which is at least an order of magnitude larger than clinically relevant concentrations (16). Propofol 10 µM was ineffective and agreed with the present data. Thus, VR1 receptors are unlikely to be a major target for IV anesthetics, possibly indicating a dichotomy in responsiveness between classical GABAA type and trp receptors

In conclusion, we suggest that local anesthetics may act as VR1 receptor antagonists, although their site or sites of action remain to be determined.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Caterina MJ, Leffler A, Malmberg AB, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000; 288: 306–13.[Abstract/Free Full Text]
  2. Johanek LM, Heitmiller DR, Turner M, et al. Cannabinoids attenuates capsaicin-evoked hyperalgesia through spinal and peripheral mechanisms. Pain 2001; 93: 303–15.[Web of Science][Medline]
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  5. Kohane DS, Kuang Y, Langer R, et al. Vanilloid receptor agonists potentiate the in vivo local anesthetic activity of percutaneously injected site 1 sodium channel blockers. Anesthesiology 1999; 90: 524–34.[Web of Science][Medline]
  6. Franks NP, Lieb WR. Molecular and cellular mechanisms of general anaesthesia. Nature 1994; 367: 607–14.[Medline]
  7. Jerman JC, Brough SJ, Prinjha R, et al. Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology. Br J Pharmacol 2000; 130: 916–22.[Web of Science][Medline]
  8. Sprague J, Harrison C, Rowbotham DJ, et al. Temperature-dependent activation of recombinant rat vanilloid VR1 receptors expressed in HEK293 cells by capsaicin and anandamide. Eur J Pharmacol 2001; 423: 121–5.[Web of Science][Medline]
  9. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440–9.[Abstract/Free Full Text]
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  11. Vedantham V, Cannon SC. The position of the fast-inactivation gate during lidocaine block of voltage-gated Na+ channels. J Gen Physiol 1999; 113: 7–16.[Abstract/Free Full Text]
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Accepted for publication January 28, 2003.




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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