Inhibitory effects of intravenous anaesthetic agents on K+-evoked norepinephrine and dopamine release from rat striatal slices: possible involvement of P/Q-type voltage-sensitive Ca2+ channels

K. Hirota1, M. Kudo1, T. Kudo1, A. Matsuki1 and D. G. Lambert2

1Department of Anesthesiology, University of Hirosaki School of Medicine, Hirosaki 036-8562, Japan. 2University Department of Anaesthesia and Pain Management, Leicester Royal Infirmary, Leicester LE1 5WW, UK*Corresponding author

Accepted for publication: March 7, 2000


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The role of the voltage-sensitive Ca2+ channel (VSCC) as a target for anaesthetic action remains controversial. In this study we characterized the VSCC subtypes involved in K+-evoked norepinephrine and dopamine release from rat striatal slices and used this model system to examine the effects of a range of i.v. anaesthetics on release. Nifedipine (L-channel-selective), {omega}-conotoxin GVIA (N-channel-selective), {omega}-agatoxin IVA (P-channel-selective), {omega}-conotoxin MVIIC (P/Q-channel-selective) and Cd2+ (non-selective), along with alphaxalone, propofol and ketamine, were used in various combinations. {omega}-Agatoxin IVA, {omega}-conotoxin MVIIC and Cd2+ fully (100%) inhibited norepinephrine and dopamine release. Clinically achievable concentrations of alphaxalone inhibited norepinephrine and dopamine release, with concentrations producing 25 and 50% inhibition (IC25 and IC50) of the maximum of 2.1 and 7.8 µM respectively for norepinephrine and 2.9 and 7.2 µM for dopamine. The effects of propofol were observed at the top of the clinical range and those of ketamine exceeded this range. In addition, IC50 values for alphaxalone in the presence and absence of nifedipine and {omega}-conotoxin GVIA did not differ from the control. Our data suggest that clinically achievable concentrations of alphaxalone and propofol inhibit norepinephrine and dopamine release, which is mediated predominantly through P/Q-type VSCCs, suggesting a role for these channels in anaesthetic action.

Br J Anaesth 2000; 85: 874–80

Keywords: anaesthetics, i.v.; rat


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Since Nowycky, Fox and Tsien classified neuronal voltage-sensitive Ca2+ channels (VSCCs) into types T, L and N, at least six classes of VSCC have been described (T, L, N, P, Q, R).1 It is clear that Ca2+ has an important role to play in neuronal signalling as this divalent cation contributes to the regulation of neuronal excitability and neurotransmitter release predominantly via the action of VSCCs.2 Of these, the L-type VSCC is located predominantly on neuronal cell bodies, is sensitive to dihydropyridines (DHPs) and may modify neurotransmitter release under certain circumstances.3 N-type VSCCs are distributed widely in the nervous system, are sensitive to {omega}-conotoxins and are involved in the regulation of neurotransmitter release.2 4 P-type VSCCs are also widely distributed in the central nervous system and account for 80% of the VSCCs present in mammalian nerve terminals.5 This type of channel is involved in central synaptic transmission58 and is blocked by {omega}-agatoxin IVA. Q-channels are closely related to the P-channels, but display lower sensitivity to {omega}-agatoxin IVA (although this toxin does block Q-channels at higher concentrations).

The molecular target site(s) for anaesthetic action remains to be determined. Whilst it is generally accepted that the GABAA receptor represents an important target site,911 there is evidence for depressed excitatory transmission12 13 and actions at VSCCs.14 15 We have previously shown that a range of i.v. anaesthetic agents (with the exception of ketamine) inhibit the binding of DHPs to L-VSCC and that there is a significant correlation between binding affinity and the peak plasma concentrations seen during anaesthesia.16 Whilst radioligand binding studies give important information about whether an interaction occurs, these studies give little indication about the functional consequences of such an interaction. Clearly, more functional studies are required.

This study had two main aims: (i) to define the VSCC subtype(s) controlling norepinephrine and dopamine release from rat striatal slices, and (ii) to examine the interaction of i.v. anaesthetic agents on this response in a more intact slice model. We examined the effects of nifedipine (L-channel-selective), {omega}-conotoxin GVIA (N-channel-selective), {omega}-agatoxin IVA (P-channel-selective), {omega}-conotoxin MVIIC (P/Q-channel-selective) and Cd2+ (non-selective) on K+-evoked norepinephrine and dopamine release from rat striatal slices. We also examined the effects of the i.v. anaesthetic agents alphaxalone, propofol and ketamine on these responses. We should emphasize that we do not claim that inhibition of norepinephrine and dopamine release from the striatum is responsible for the anaesthetic state (although this is possible); we used this model system to study any role of VSCC in the actions of three i.v. anaesthetic agents.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Materials and stock solutions
{omega}-Conotoxin GVIA, {omega}-agatoxin IVA and {omega}-conotoxin MVIIC were purchased from the Peptide Institute (Osaka, Japan). Nifedipine was purchased from Wako (Osaka, Japan), pargyline, nomifensin and i.v. anaesthetic agents were from Sigma-Aldrich Japan (Tokyo, Japan), and HEPES from Dojin Laboratories (Kumamoto, Japan). All other chemicals used were of the highest quality available. Stock solutions were made as follows: pure propofol (100 mM stock in dimethyl sulphoxide, DMSO); alphaxalone and nifedipine (50 mM stock in DMSO), ketamine and CdCl2 (100 mM stock in distilled water); and {omega}-conotoxin GVIA, {omega}-conotoxin MVIIA and {omega}-agatoxin IVA (10–4 M in distilled water). All agents were then diluted in Krebs–Ringer bicarbonate buffer; the highest anaesthetic concentration used was limited by agent solubility.

Preparation of slices
Male Wistar rats (250–300 g) were decapitated and their brains were quickly removed and immersed in ice-cold Krebs–Ringer bicarbonate buffer solution (KRBS) of the following composition (mM): NaCl 133, KCl 4.8, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.5, glucose 11.1, HEPES 10 (pH 7.4). Striatal tissue was dissected and cross-chopped using a tissue chopper to produce slices of 350x350 µm. The slices were then washed three times in ice-cold KRBS and transferred (1 ml aliquots of slices, equivalent to about 6 mg protein) to polypropylene tubes. Striatal slices from one rat were used for one experiment (i.e. one concentration– response curve was constructed for each agent).

Norepinephrine and dopamine release experiments
After the supernatant had been discarded, the slices were resuspended in 1 ml of fresh KRBS and incubated for 10 min at 37°C. This procedure was repeated to obtain a stable baseline. Immediately after this second incubation, the slices were resuspended (1 ml KRBS) and incubated for 0–9 min in the absence (basal release) and presence (evoked release) of 40 mM KCl. In some experiments, slices were incubated for a fixed time of 6 min with KRBS containing 0–70 mM KCl in order to obtain a K+-evoked release concentration–response curve. All buffers used in release studies contained the monoamine oxidase inhibitor pargyline (10 µM), and the reuptake inhibitor nomifensin (10 µM) to amplify the release signal by preventing amine breakdown and reuptake respectively. In addition, for samples containing K+, an equal concentration of Na+ was removed.

Effects of VSCC blockers and i.v. anaesthetics on K+-evoked norepinephrine and dopamine release
Slices were prepared and incubated as above. They were resuspended in 1 ml KRBS containing nifedipine (10–11 to 10–6 M), {omega}-conotoxin GVIA (10–11 to 10–6 M), {omega}-agatoxin IVA (3x10–9 to 10–6 M), {omega}-conotoxin MVIIC (3x10–9 to 10–6 M) and Cd2+ (10–6 to 3x10–4 M), alphaxalone (10–8 to 10–4 M), propofol (10–8 to 10–3 M) and ketamine (10–8 to 10–3 M) in various combinations and incubated for a further 10 min at 37°C. This procedure was repeated. Six minutes of basal sampling was then performed before the slices were finally exposed to 40 mM K+ for 6 min in the continued presence of the test agents as appropriate.

To examine whether the inhibitory effects of alphaxalone on norepinephrine and dopamine release are mediated mainly via P and Q-VSCCs, KRBS contained various concentrations of alphaxalone and 40 mM K+ with or without nifedipine (10–6 M) and {omega}-conotoxin GVIA (10–6 M).

Measurement of norepinephrine and dopamine release
Monoamine contents in the release samples were determined directly by high-performance liquid chromatography with an electrochemical detector (Model 5100A, ESA Coulochem) as described by Takeda and colleagues.17 Aliquots (20 µl) of acidified (perchloric acid) release samples were injected onto a reverse-phase column (C18, 4.6x150 mm; MC Medical, Tokyo, Japan). For each experimental run, a standard curve for norepinephrine and dopamine was constructed (using 3,4-dihydroxybenzylamine as the internal standard). The intra-assay coefficient of variation was 3.3% for norepinephrine and 6.5% for dopamine.

Calculations and data analysis
All data are presented as mean (SEM) (n). The concentrations of VSCC blockers and anaesthetic agents producing 25% (IC25) and 50% (IC50) inhibition were estimated from individual curves by non-linear regression analysis (Graphpad-Prism v. 2.0). When appropriate, statistical analysis was by repeated measures ANOVA and the unpaired t-test for intra- and inter-group comparison respectively. P<0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Norepinephrine and dopamine release evoked by K+ was time-dependent, reaching a maximum some 6–7 min after stimulation (Table 1). In addition, K+ produced concentration-dependent release of norepinephrine and dopamine, with pEC50 values of 1.41 (0.02) (38.8 mM) and 1.46 (0.03) (34.9 mM) respectively (Fig. 1).


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Table 1 Time course of K+-evoked norepinephrine and dopamine release. Mean of two measurements, expressed as pmol tube–1; each tube contained 1 ml of sample and about 6 mg protein
 


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Fig 1 K+-evoked norepinephrine and dopamine release from rat striatal slices is concentration-dependent. Concentrations producing 50% of the maximum response (IC50) were 38.8 and 34.9 mM for norepinephrine and dopamine respectively. Data are mean (pg ml–1) and SEM (n=4).

 
Effects of VSCC blockers on neurotransmitter release
The non-selective VSCC antagonist Cd2+ completely abolished norepinephrine and dopamine release (Fig. 2A). In addition, selective VSCC antagonists also reduced release to varying degrees (Fig. 2BE, Table 2). L-VSCC blockade with nifedipine (Fig. 2B) produced only a 15–20% inhibition of release (P<0.01). N-VSCC blockade (Fig. 2C) also produced a small but significant inhibition of release (P<0.01). In contrast, blockade of P/Q VSCC (Fig. 2D and E) produced a marked inhibition of release.



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Fig 2 Effects of Ca2+ channel blockers Cd2+ (A, non-selective), nifedipine (B, L type), {omega}-conotoxin GVIA (C, N type), {omega}-agatoxin IVA (D, P type), and {omega}-conotoxin MVIIC (E, P/Q type) on K+-evoked norepinephrine and dopamine release. Data are mean and SEM (n=6).

 

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Table 2 pIC50 (mean IC50, maximum inhibition) of VSCC antagonists for K+-evoked norepinephrine and dopamine release. Data are mean (SEM)
 
Effects of i.v. anaesthetics on neurotransmitter release
Alphaxalone produced concentration-dependent and full inhibition of norepinephrine and dopamine release (Fig. 3, Table 3). In addition, dose–response curves for the inhibition of norepinephrine and dopamine release by alphaxalone in the presence and absence of L- and N-type VSCC blockers coincided (Fig. 4). The pIC50 values (mean IC50) for the inhibition of norepinephrine and dopamine release by alphaxalone in the presence of nifedipine and {omega}-conotoxin GVIA (5.11 (0.11) (7.8 µM) and 5.22 (0.08) (6.1 µM) respectively) were similar to those in the absence of nifedipine and {omega}-conotoxin GVIA (Table 3).



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Fig 3 Effects of i.v. anaesthetics (alphaxalone, ketamine and propofol) on K+-evoked release of norepinephrine (A) and dopamine (B). Data are mean and SEM (n=5 or 6).

 

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Table 3 pIC25 and pIC50 (mean IC25 and IC50) of i.v. anaesthetics for K+-evoked norepinephrine and dopamine release. Data are mean (SEM). *Plasma concentration of anaesthetic agent during anaesthesia (µM).
 


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Fig 4 Concentration–response curves for alphaxalone inhibition of K+-evoked release of norepinephrine (A) and dopamine (B) in the presence and absence of L- and N-type Ca2+ channel blockers. Data are mean and SEM (n=6).

 
The inhibitory effects of propofol were weaker than those of alphaxalone, and these did not saturate at the highest concentration used. Curve fits for propofol and ketamine were poor because of this lack of saturation, and therefore the estimated IC25 and IC50 values should be interpreted with caution. Notwithstanding, the estimated IC25 values for the inhibition of norepinephrine and dopamine release by propofol were close to the total plasma concentrations seen during anaesthesia (although the free concentration would be lower) (Fig. 3, Table 3). In contrast, the effects of ketamine on norepinephrine and dopamine release occurred at concentrations in excess of those encountered clinically (Fig. 3, Table 3).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Consistent with previous reports,18 19 neurotransmitter release evoked by K+ (depolarization) was time- and concentration-dependent. Although Harvey and colleagues19 showed that the response to K+ differed for each neurotransmitter, dopamine and norepinephrine showed similar responses in the present study. Harvey and colleagues19 demonstrated that internal calcium is unlikely to be involved in the release of these catecholamines from brain slices. It was concluded that K+-evoked dopamine and norepinephrine release are mediated predominantly by VSCCs, and the same argument can be applied to our data. However, there are preparations in which the release of internal stored Ca2+ is capable of supporting release, albeit to a lesser extent. For example, we have shown that carbachol (acting via the M3 muscarinic receptor) is capable of eliciting norepinephrine release from SH-SY5Y human neuroblastoma cells in the absence of external Ca2+, implying that the release of stored Ca2+ evokes transmitter release.20

Nifedipine, an L-type VSCC blocker, inhibited dopamine and norepinephrine release from rat striatal slices by 15–20%. The IC50 of nifedipine is consistent with the Ki of this agent for the displacement of [3H]PN200–110 binding to rat striatal membranes reported previously.21 The release of dopamine and norepinephrine from rat brain slices has been reported to be mediated partially through N-type, but more markedly through P/Q-type VSCCs.4 18 19 22 23 In agreement with these reports, the present study also showed that the inhibition of catecholamine release by the range of selective VSCC blockers studied was similar to that reported previously.4 19 23 However, combined inhibition produced by the VSCC blockers used was in excess of 100% (e.g. for norepinephrine release, nifedipine inhibited by 20%, {omega}-conotoxin GVIA by 35% and {omega}-agatoxin IVA by 88%; the combined inhibition was 143%) and {omega}-conotoxin MVIIC inhibited norepinephrine and dopamine release completely at 1 µM. Therefore, these toxins at high concentrations produce less selective inhibition of VSCCs. This is in agreement with previous observations.4 19

A unifying target site for anaesthetic action remains elusive. However, the GABAA receptor represents an important target with which all anaesthetic agents so far examined (excepting ketamine, nitrous oxide and xenon) interact. Anaesthetic agents potentiate the GABA-induced chloride conductance, leading to hyperpolarization and depressed neurotransmission.911

There is evidence to support a role for VSCCs as an additional cellular target for anaesthetic agents.14 15 L-VSCC blockers such as verapamil and nitrendipine augment general anaesthetic potencies in laboratory animals.24 26 We have also shown that i.v. anaesthetic agents (except ketamine) inhibit the binding of DHPs to L-VSCC, and there is a significant correlation between K25 and K50 for inhibition of DHP binding by anaesthetics and the peak plasma concentrations seen during anaesthesia.16 In addition, several reports have show that clinically relevant concentrations of general anaesthetics inhibit DHP-insensitive Ca2+ currents.2729 However, we have recently questioned the role of this channel in man.30

A range of anaesthetic agents have been reported to inhibit excitatory synaptic transmission, and this may be via block of entry through receptor-operated and/or voltage-sensitive Ca2+ channels.14 However, we have recently reported that propofol and thiopental inhibit glutamate release from rat cerebrocortical slices in a bicuculline-sensitive fashion, indicating that these anaesthetics are inhibiting release via an action at GABA receptors [31]. This inhibition may lead to some components of general anaesthesia, such as loss of consciousness and analgesia.

In the present study, alphaxalone produced concentration-dependent inhibition of norepinephrine and dopamine release, with IC25 and IC50 values occurring at clinically achievable concentrations.14 In addition, propofol also produced concentration-dependent inhibition of norepinephrine and dopamine release, with IC25 values approaching those achieved clinically.16 We have previously argued in favour of the use of IC25 for Ca2+ responses,14 16 as relatively small increases in intracellular Ca2+ are capable of eliciting release. Although the total plasma concentration would be markedly reduced by protein binding, it should be noted that propofol is concentrated some eight-fold in the rat brain.32 We therefore suggest that neuronal VSCCs represent an additional target for the action of alphaxalone and possibly propofol. The effects of ketamine observed in this study occurred at concentrations in excess of those encountered clinically,33 and we therefore feel that the actions of this atypical anaesthetic34 do not involve VSCCs. Moreover, it is well known that this anaesthetic agent acts at the NMDA–glutamate receptor.13

As the action of alphaxalone occurred in the clinically relevant range, we investigated this agent further. There were no significant differences in the IC50 values of alphaxalone for K+-evoked norepinephrine and dopamine release in the presence and absence of nifedipine and {omega}-conotoxin GVIA. These findings suggest that the inhibition by alphaxalone of norepinephrine and dopamine release may not be via the L/N-VSCC, implicating P/Q-type channels. However, Kameyama and colleagues35 reported differential effects of isoflurane on VSCCs of the L, N and P/Q types, as the levels of inhibition of these channel currents produced by 0.5 mM isoflurane were 60, 35 and 15% respectively. This discrepancy may be a result of differences in the type of anaesthetic agent (steroid vs volatile anaesthetic), as alphaxalone and isoflurane produce different clinical anaesthetic effects. In addition, there are quantitative differences between electrically evoked and K+-evoked neurotransmitter release.22

In summary, the present study shows that clinically achievable concentrations of alphaxalone significantly inhibit K+-evoked norepinephrine and dopamine release from rat striatal slices, and this may be mediated by P/Q-type VSCCs. However, further studies examining bicuculline sensitivity need to be performed to determine any contribution of GABAA receptor activation to these responses.


    Acknowledgements
 
This work was supported in part by grant-in-aid for scientific research (No. 09470323) from the Ministry of Education, Science and Culture, Japan.


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 Introduction
 Methods
 Results
 Discussion
 References
 
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