1University Department of Anaesthesia, Leicester Royal Infirmary, Leicester, LE1 5WW, UK. 2Oregon Health Sciences University, Portland, OR, USA
Accepted for publication: June 9, 2000
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Abstract |
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Br J Anaesth 2000; 85: 7406
Keywords: anaesthetics local; measurement techniques, radioligand binding; metabolism, cAMP
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Introduction |
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Local anaesthetic agents produce a use dependent block of voltage dependent Na+ channels, and hence, reduce axonal conduction.8 Opioid receptors are classified as µ, and
and all have been cloned and expressed in a variety of cells and display pharmacology consistent with the receptors previously identified. Opioid receptor activation activates an inwardly rectifying K+ channel (Kir-hence promoting an efflux of K+) and closes voltage sensitive Ca2+ channels resulting in membrane hyperpolarization, reduced Ca2+ influx, and hence, reduced neurotransmission. Adenylyl cyclase is also inhibited leading to reduced cAMP formation.914 This reduced cAMP formation is often measured as a biochemical index of opioid receptor activation but may also be involved in reduced neuronal firing via an interaction with the inwardly rectifying K+ channel (Ih).15
The mechanism and site(s) of opioid-local anaesthetic interaction are yet to be fully described. Several potential sites are possible. These include: changes in opioid pharmacokinetics (i.e. changes in tissue pH) produced by local anaesthetics;5 potentiation of the inhibitory effect of opioids on neurotransmitter release via modulation of second messenger systems such as adenylyl cyclase;12 14 16 or actions at voltage sensitive Ca2+ channels.17 18 A direct interaction of local anaesthetics with opioid receptors has also been suggested.7
In this study, we have tested the latter hypothesis, that local anaesthetics interact with opioid receptors and that this may modulate the formation of cAMP. In order to avoid the interpretation problems associated with cell and tissue studies of a heterogeneous population of endogenous receptors, we have utilized Chinese hamster ovary (CHO) cells expressing recombinant µ-, -, and
-opioid receptors (CHO-µ,
, and
, respectively).
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Materials and methods |
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Membrane preparation and cell culture
CHO-µ, ,
cells1921 and untransfected CHO wild-type (CHO-wt) cells (i.e. cells not expressing the plasmid that encodes the receptor of interest and, therefore, acting as negative controls) were grown for experimentation in Hams F12 medium supplemented with penicillin 100 iu ml1, streptomycin 100 µg ml1, fungizone 2.5 µg ml1 and fetal calf serum 10%. (Stock cultures also contained G418 200 µg ml1.) Cultures were maintained at 37°C in 5% CO2/humidified air at 37°C, fed every 23 days and passaged every 7 days. Experiments were performed at days 57 after subculture. All cells were harvested for use by the addition of saline 0.9% containing HEPES (10 mM)/EDTA (0.02%). Cells were homogenized at 4°C using a tissue Tearor (setting 5.5x30-s bursts) in 50 mM TrisHCl buffer (pH 7.4). The homogenate was centrifuged at 18 000 xg for 10 min and the pellet resuspended in TrisHCl buffer. This procedure was repeated twice more. Membranes were prepared and used fresh daily.
[3H]DPN binding
The binding of the non-selective opioid receptor antagonist [3H]DPN was performed in 1 ml volumes of TrisHCl buffer containing approximately 200 µg of membranes at 20°C for 90 min as described previously.22 Non-specific binding was defined in the presence of naloxone 10 µM. Following incubation, each sample was filtered (and washed) under vacuum through Whatman GF/B filters using a Brandel cell harvester. Filter retained radioactivity was extracted for at least 8 h in 4 ml of scintillation fluid. In displacement studies, the interaction of lidocaine (106 to 102 M) with µ, and
-opioid receptors, and of bupivacaine (106 to 3x103 M) and its enantiomers (S() and R(+): 106 to 103 M), procaine (106 to x102 M), prilocaine (106 to 3x102 M), and tetracaine (106 to 102 M) with µ-opioid receptor were determined by displacement of
0.5 nM [3H]DPN.
Measurement of cAMP formation
Whole cells (CHO-µ, ,
and wt) suspended in 0.3 ml KrebsHEPES buffer, pH 7.4 were incubated in the presence of isobutylmethylxanthine (1 mM) with or without (for the basal) forskolin (10 µM) at 37°C for 15 min. To obtain a concentration response curve for lidocaine inhibition of cAMP formation, cells were incubated additionally with or without lidocaine (105 to 3x102 M). In order to determine any naloxone-sensitivity of lidocaine inhibition of cAMP formation, CHO-µ,
or
cells were incubated with lidocaine 2 mM in the presence or absence of naloxone 10 µM. In addition some cells were co-incubated with lidocaine (300 µM) and subtype selective opioid agonists: (D-Ala2, MePhe4, Gly(ol)5) enkephalin (DAMGO: 100 nM) for µ and spiradoline (2 nM) for
. Reactions were terminated by the addition of 20 µl HCl (10 M), 20 µl NaOH (10 M) and 180 µl Tris buffer (1 M, pH 7.4). The concentration of cAMP was measured in the supernatant following centrifugation (13 000 r.p.m./2 min) using a specific protein binding assay as described previously.22
Statistical analysis
All data are expressed as mean (SEM). The log concentration of displacers producing 50% displacement of specific binding (IC50) and the concentration of local anaesthetic producing 50% maximum inhibition of forskolin stimulated cAMP formation (IC50) were obtained by computer assisted curve fitting (GRAPHPAD-PRISM). IC50 values from binding experiments were additionally corrected for the competing mass of [3H]DPN according to Cheng and Prusoff23 to yield the inhibitor constant (Ki). Further terminology follows IUPHAR recommendations where Bmax is defined as the maximum specific binding of a ligand determined in a radioligand binding assay (an estimate of the number of receptors), and Kd is the equilibrium dissociation constant (calculated as ligand concentration at 0.5 Bmax). Additivity data are analysed using an unpaired t-test with P<0.05 considered to represent a significant difference.
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Results |
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Discussion |
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Local anaesthetic agents have been shown to interact with a number of differing receptor systems including ligand gated ion channels and G-protein coupled receptors. For example, the GABAA receptor27 and nicotinic receptor28 are ligand gated ion channel coupled receptors whose activity is inhibited by local anaesthetics. Interestingly, in the latter study, glutamate, NMDA and AMPA receptors were unaffected.28 Some examples of the interaction of local anaesthetic agents with G-protein coupled receptors are shown in Table 1.2931 For this receptor system there does not appear to be any significant correlation with relative conduction blocking potency but the rank order potency at the µ opioid receptor follows the hydrophobicity ranking.
A possible target for the interaction of local anaesthetics and opioids is the opioid receptor and the elements of the signal transduction cascade associated with this receptor, i.e. adenylyl cyclase, voltage sensitive Ca2+ channels and inwardly rectifying K+ channels. Tejwani and colleagues7 reported that bupivacaine inhibited the specific binding of a range of radioligands to µ-receptors but surprisingly increased binding to - and
-receptors. However, in a radioligand binding study, Fraser and Fowler32 reported that local anaesthetics did not interact with
-opioid receptors. The discrepancy between these and our study most likely results from the concentration of local anaesthetic used. In the studies of Tejwani and colleagues7 and Fraser and Fowler32 bupivacaine concentration of less than 10 µM were used. The present data indicates that significant inhibition of radioligand binding to opioid receptors requires bupivacaine concentrations in excess of 10 µM (Ki, 161 µM).
In a review article, Franks and Lieb33 suggested that stereoselectivity may prove to be one of the most powerful guides to in vitro targets relevant to anaesthesia. In the present study (with the amount of bupivacaine isomers available to us) our data give an indication of some stereoselectivity for bupivacaine at µ- and - but probably not
-opioid receptors (Fig. 3) with the R(+) isomer being more potent than the S() isomer. However, this stereoselectivity is the reverse of that observed for neuronal Na+ channels (S() more potent than (R(+))34 and we have no explanation for this discrepancy. Despite the conclusions of Fraser and Fowler32 to the contrary, our data suggest that the µ- and
-opioid receptors may represent a target for local anaesthetic agents and a site at which the clinically observed local-opioid interaction may occur. In this study, we probed the functional consequences of this interaction further by studying the effects of opioids and lidocaine on the formation of cAMP.
Opioid receptor agonists inhibit adenylyl cyclase thus inhibiting cAMP formation through opioid receptors and this may give clues as to an interaction with Gi coupled voltage sensitive Ca2+ channels and Kir. In addition, Wang and colleagues35 reported that cAMP mediated µ and but not
opioid analgesia, based on the reversal of µ and
-mediated analgesia by intrathecal administration of dibutyl-cAMP (i.e. artificially elevating cAMP). In the present study lidocaine inhibited forskolin-stimulated cAMP formation in a concentration-dependent and naloxone insensitive manner indicating that this inhibitory action on adenylyl cyclase was not mediated via opioid receptors. This was further confirmed in untransfected CHO cells where lidocaine also inhibited cAMP formation with an essentially identical pIC50 when compared with CHO-
, µ and
cells. These data are also consistent with the observed direct interaction of local anaesthetics with the catalytic subunit of adenylyl cyclase leading to reduced cAMP formation.16 Thus, whilst the formation of cAMP is, as would be expected for opioids, inhibited by lidocaine, this response does not represent agonist action at the opioid receptor. If local anaesthetic agents and opioid receptor activation inhibit cAMP formation by different pathways then it might be reasonable to assume that the combination of local and opioid would produce an additive inhibition. This was clearly not the case (Table 3). Indeed we have found a suggestion of an inhibitory action of lidocaine on
(and possibly µ) receptor signaling. We cannot explain the lack of a positive interaction between these two agents at the level of cAMP formation.
Our data suggest that the interaction between local anaesthetic agents and opioids in the clinical setting does not result from an interaction with opioid receptor signaling (i.e. at the cellular level). It is well known that opioids inhibit cAMP formation, close voltage sensitive Ca2+ channels and activate inwardly rectifying K+ channels. These combined actions are likely to reduce neurotransmission. It is also well known that local anaesthetic agents inhibit voltage sensitive Na+ channels to reduce axonal conduction. Na+ channel blockade with local anaesthetics produces analgesia. It is worthy of mention that a range of anticonvulsants with analgesic activity also appear to inhibit voltage sensitive Na+ channels. For example, carbamazepine36 and gabapentin37 block Na+ channels and are also useful in the pain clinic. The actions of lamotrigine are equivocal where Na+ channel block36 and facilitation of C-fibre responses38 have also been reported.
We therefore conclude that the clinical interaction between local anaesthetic agents and opioids may occur as a combination of reduced axonal conduction and neurotransmission in the spinal cord and an interaction at the cellular level is unlikely.
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Acknowledgements |
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Footnotes |
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References |
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