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Effects of intravenous and local anesthetic agents on -conotoxin MVII_A binding to rat cerebrocortex

Kazuyoshi Hirota, MD^*, and David G. Lambert, PhD

^* From the University Department of Anaesthesia and Pain Management, Leicester Royal Infirmary,
Leicester LE1 5WW, UK, and Department of Anesthesiology University of Hirosaki School of Medicine, Hirosaki, Japan.

Address correspondence to: Dr. D.G. Lambert. Phone: +44-116-258-5291; Fax: +44-116-285-4487; E-mail: DGL3{at}le.ac.uk

	Abstract

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Purpose: The cellular target site(s) for anesthetic action remain controversial. In this study we have examined any interaction of iv anesthetics (thiopental, pentobarbital, ketamine, etomidate, propofol, alphaxalone), local anesthetics (lidocaine, prilocaine, procaine and tetracaine), and the non anesthetic barbiturate, barbituric acid with the -conotoxin MVII_A binding site on N-type voltage sensitive Ca²⁺ channels in rat cerebrocortical membranes.

Methods: [¹²⁵I] -conotoxin MVII_A binding assays were performed in 0.5 ml volumes of Tris.HCl buffer containing BSA 0.1% for 30 min at 20°C using fresh cerebrocortical membranes (5 µg of protein). Non-specific binding was defined in the presence of excess (10^–8 M) -conotoxin MVII_A. The interaction of iv (alphaxolone, etomidate, propofol, pentobarbitone, ketamine and thiopentone), local (lidocaine, prilocaine, procaine and tetracaine) anesthetics and barbituric acid was determined by displacement of [¹²⁵I] -conotoxin MVII_A (~1pM).

Results: The binding of [¹²⁵I] -conotoxin was concentration-dependent and saturable with B_max and K_d of 223 ± 15 fmol/mg protein and 2.13 ± 0.14 pM, respectively. Unlabelled -conotoxin MVII_A displaced [¹²⁵I] -conotoxin MVII_A yielding a pK_d of 11.04 ± 0.04 (9.2 pM). All iv and local anesthetics at clinically relevant concentrations did not show any interaction with the -conotoxin MVII_A binding site.

Conclusion: The present study suggests that -conotoxin MVII_A binding site on N-type voltage sensitive Ca²⁺ channels may not be a target for iv and local anesthetic agents.

	Introduction

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THE role of voltage sensitive Ca²⁺ channels (VSCC) in anesthesia is controversial.^1,2 We have shown that intravenous³ and local⁴ anesthetics interact with dihydropyridine binding sites on L-type VSCC. Moreover, there was a correlation between anesthetic potency and interaction with the dihydropyridine binding sites.^3,4 Despite the observation that central transmission is not usually under the control of L-VSCC these data might implicate L-VSCC as anesthetic target sites.

N-type VSCC are distributed widely in the nervous system and play a far more important role in the control of neurotransmitter release.² Electrophysiological (functional) studies suggest that clinically relevant concentrations of propofol,⁵ several barbiturates⁶ and local anesthetic agents⁷ significantly depress -conotoxin sensitive N-channel currents. The precise site of this interaction is unclear.

As part of a broader pharmacological examination of the role of VSCC in anaesthesia (and to allow comparison L-VSCC data) we have examined whether a range of intravenous and local anesthetic agents interact with -conotoxin binding sites on N-type VSCC. -Conotoxin VII_A was used as the binding is fully reversible and ideally suited to radioligand binding studies.

	Methods

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Tissue preparation
Female Wistar rats (250-300g) were stunned then decapitated. The brain was rapidly removed, the cerebrocortex detached from its internal structures, placed in 50 mM Tris.HCl, pH7.4 at 4°C, and homogenized using an ultra turrax (T25). The resulting homogenate was centrifuged at 18000 g for 10 min and the pellet resuspended in Tris.HCl buffer. This procedure was repeated three times.

Measurement of [¹²⁵I] -conotoxin MVII_A binding.
All binding assays were performed in 0.5 ml volumes of Tris.HCl buffer containing 0.1% bovine serum albumin for 30 min at 20°C using 5 µg fresh membranes. Saturation analyses to determine the equilibrium dissociation constant (K_d) and the maximal binding capacity (B_max) were performed using increasing concentrations of [¹²⁵I] -conotoxin MVII_A (0.1-10 nM). Non-specific binding was defined with 10^–8M -conotoxin MVII_A. Bound and free radioactivity were separated using a Brandell cell harvester onto Whatman GFB filters (treated with 0.1% polyethylenimine) with bound estimated using a -counter.

In order to determine if an agent interacted with -conotoxin binding sites on N-VSCC, these were labeled with a fixed concentration of [¹²⁵I] -conotoxin MVII_A. Increasing concentrations of unlabeled displacers (anesthetic agents) were added, and displacement of [¹²⁵I] -conotoxin MVII_A binding indicates an interaction at the same site as the radiolabel. [¹²⁵I] -conotoxin MVII_A (~1pM) was incubated with various concentrations of the following unlabelled intravenous anesthetic agents: thiopental, pentobarbital, ketamine, etomidate, propofol and alphaxalone, and local anesthetic agents: lidocaine, prilocaine, procaine and tetracaine. A non-anesthetic barbiturate, barbituric acid was also included. Unlabeled -conotoxin MVII_A was included as a positive control. Agents were dissolved in Tris.HCl buffer from stocks as follows; thiopental (100 mM stock in 0.1M NaOH), pentobarbital and barbituric acid (50 mM stock in distilled water), propofol (100 mM stock in DMSO), alphaxalone (50 mM stock in DMSO), etomidate (50 mM stock in 0.1M HCl), ketamine and local anesthetic agents (500 mM stock in distilled water), and -conotoxin MVII_A (10^-6M in distilled water). Anesthetic concentration used was limited by solubility.

Data analysis
B_max and K_d were obtained from Scatchard transformation of the specific binding data. The log concentration unlabelled conotoxin producing 50% displacement of labelled conotoxin binding (i.e., isotope dilution), pK_d was obtained by computer assisted curve fitting (GraphPad-PRISM). All data are expressed as mean ± SEM, n refers to the number of individual full saturation or displacement curves constructed.

	Results

TOP Abstract Introduction Methods Results Discussion References

 
The binding of [¹²⁵I] -conotoxin was concentration-dependent and saturable with B_max and K_d of 223 ± 15 fmol•mg^-1 protein and 2.13 ± 0.14 pM, respectively. Unlabelled -conotoxin MVII_A displaced [¹²⁵I] -conotoxin MVII_A yielding a pK_d of 11.04 ± 0.04 (9.2 pM), in reasonable agreement with the value obtained by direct saturation. These K_d and B_max values for [¹²⁵I] -conotoxin MVII_A binding were similar to those previously reported by Yamada et al.⁸ More importantly, all anesthetic agents at clinically relevant concentrations failed to displace [¹²⁵I] -conotoxin MVII_A (Figure).

View larger version (15K): [in this window] [in a new window]   FIGURE Unlabeled -conotoxin MVIIA (A) but not Iv (B) or local (C) anesthetic agents at clinically relevant concentrations displaced [125I] -conotoxin MVIIA binding to N-type voltage sensitive Ca2+ channels. Data are mean ± SEM (n=6) and expressed as displacement where the curves were corrected for the competing mass of [125I] -conotoxin MVIIA.

	Discussion

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In the search for a common target site for anesthetic agents, voltage and ligand gated ion channels have been extensively studied. It has been suggested that GABA_A receptors and voltage sensitive Na⁺ channels may be the most important target sites for general and local anesthesia, respectively.^1,9 However, Ca²⁺ is also likely to play an important role in the mechanism of general and local anesthesia, as this cation regulates neuronal excitability and neurotransmitter release through VSCC.²

Several reports^5–7 suggest that intravenous and local anesthetics at clinical concentrations inhibit N-VSCC. These data implicate N-VSCC in the mechanism of anesthesia. However, in the present study, clinically relevant concentrations of intravenous and local anesthetic agents did not displace -conotoxin MVII_A binding to N-VSCC. Nacif-Coelho and colleagues¹⁰ failed to report any involvement of N-VSCC in the hypnotic response to dexmedetomidine as the hypnotic effects of this agent were not modified by -conotoxin MVII_A. However, nifedipine (L-VSCC blocker) and -conotoxin MVII_C (P+Q-VSCC blocker) produced a loss of righting reflex in the presence of a subhypnotic dose. The clinical significance of these data are difficult to state as this is a mechanistic study. Collectively our data suggest that -conotoxin MVII_A binding sites on N-VSCC may not be an important anesthetic target. To test this hypothesis in man is not at present feasible as there are currently no N-VSCC blockers available for clinical use.

	Acknowledgments

 
Supported in part by a project grant from the British Journal of Anaesthesia.

Accepted for publication February 4, 2000.

	References

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1 Franks NP, Lieb WR. Molecular and cellular mechanisms of general anaesthesia. Nature 1994; 367: 607–14.[Medline]

2 Hirota K, Lambert DG. Voltage sensitive Ca²⁺ channels and anaesthesia (Editorial). Br J Anaesth 1996; 76: 344–6.[Free Full Text]

3 Hirota K, Lambert DG. I.v. anaesthetic agents inhibit dihydropyridine binding to L-type voltage-sensitive Ca²⁺ channels in rat cerebrocortical membranes. Br J Anaesth 1996; 77: 248–53.[Abstract/Free Full Text]

4 Hirota K, Browne T, Appadu BL, Lambert DG. Do local anaesthetics interact with dihydropyridine binding sites on neuronal L-type Ca²⁺ channels? Br J Anaesth 1997; 78: 185–8.[Abstract/Free Full Text]

5 Olcese R, Usai C, Maestrone E, Nobile M. The general anesthetic propofol inhibits transmembrane calcium current in chick sensory neurons. Anesth Analg 1994; 78: 955–60.[Abstract/Free Full Text]

6 Gundersen CB, Umbach JA, Swartz BE. Barbiturates depress currents through human brain calcium channels studies in Xenopus oocytes. J Pharmacol Exp Ther 1988; 247: 824–9.[Abstract/Free Full Text]

7 Sugiyama K, Muteki T. Local anesthetics depress the calcium current of rat sensory neurons in culture. Anesthesiology 1994; 80: 1369–78.[Medline]

8 Yamada S, Uchida S, Ohkura T, et al. Alterations in calcium antagonist receptors and calcium content in senescent brain and attenuation by nimodipine and nicardipine. J Pharmacol Exp Ther 1996; 277: 721–7.[Abstract/Free Full Text]

9 Strichartz GR, Berde CB. Local anesthetics. In: Miller RD (Ed.). Anesthesia, 4th ed. New York: Churchill-Livingston, 1994: 489–521.

10 Nacif-Coelho C, Correa-Sales C, Chang LL, Maze M. Perturbation of ion channel conductance alters the hypnotic response to the ₂-adrenergic agonist dexmedetomidine in the locus coeruleus of the rat. Anesthesiology 1994; 81: 1527–44.[Medline]

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