I.v. anaesthetic agents inhibit dihydropyridine binding to L-type voltage ...

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while the DHP L-channel activator, Bay K 8644, reduced the anaesthetic ... for halothane in dogs. ... high-dose pentobarbital in severe head trauma. Clinical.
British Journal of Anaesthesia 1996; 77: 248–253

I.v. anaesthetic agents inhibit dihydropyridine binding to L-type ; channels in rat cerebrocortical membranes† voltage-sensitive Ca2; K. HIROTA AND D. G. LAMBERT

Summary Previous studies have implicated the neuronal L; type voltage-sensitive Ca2 channel (VSCC) as a target site for i.v. anaesthetic agents. It is unclear if these agents interact with the L-channel ␣-subunit 1,4-dihydropyridine (DHP) binding site. In this study, we have examined the interaction of thiopentone, pentobarbitone, ketamine, etomidate, propofol and alphaxalone, and the non-anaesthetic barbiturate, barbituric acid, with the DHP binding site on rat cerebrocortical membranes. Binding assays were performed in 1-ml volumes of Tris-HCl 9 50 mmol litre 1, pH 7.4, for 90 min at room temperature containing 200 ␮g of membrane protein with [3H] PN200-110 as a radiolabelled DHP. Nonspecific binding was defined in the presence of 9 9 nifedipine 10 5 mol litre 1. The interaction of i.v. anaesthetics was determined by displacement of 9 [3H] PN200-110 0.2 nmol litre 1. All i.v. anaesthetics showed some interaction with the DHP binding site. The concentrations of anaesthetic producing 25 % inhibition of specific binding (corrected for the competing mass of [3H]PN2009 110), K25 were (␮mol litre 1): thiopentone 48 (SEM 2), pentobarbitone 95 (7), propofol 40 (2), etomidate 25 (2), alphaxalone 17 (3) and ketamine 198 (16). Barbituric acid was ineffective. With the exception of ketamine, there was a significant correlation between K25 and peak serum concentration during anaesthesia (P : 0.033) and serum concentrations on wakening (P : 0.018), suggesting that the L-channel DHP binding site may be a target for i.v. anaesthetic agents. (Br. J. Anaesth. 1996; 77: 248–253) Key words Anaesthetics i.v. lons, ion channels. lons, calcium. Model, rat.

Neuronal L-type voltage-sensitive Ca2; channels are located predominantly on neuronal cell bodies and are involved in the regulation of neurotransmitter release [1]. Moreover, L-type Ca2; channel blockers potentiate anaesthetic potency in vivo [2–6]. These findings suggest that L-type voltage-sensitive Ca2; channels may be a target site for general anaesthetic agents. Indeed, i.v. anaesthetics are known to interact with 1,4-dihydropyridine (DHP) binding sites on L-type Ca2; channels in skeletal [7] and

smooth muscle [8], and there is evidence to suggest that i.v. anaesthetics inhibit neuronal L-type Ca2; channels [9, 10]. It is still unclear, however, if i.v. anaesthetic agents interact directly with DHP binding sites on the ␣1 subunit of neuronal L-type Ca2; channels. In contrast, the interaction of volatile general anaesthetic agents is well documented [11, 12]. In this study, we examined if a range of i.v. anaesthetic agents interact with the DHP binding site (labelled with (;)-[methyl-3H]PN200-110, (;)[methyl-3H]isopropyl-4-(2,1,3-benzoadiazol-4-yl)1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3pyridinecarboxylate) on neuronal L-type Ca2; channels in rat cerebrocortical membranes.

Materials and methods TISSUE PREPARATION

Female Wistar rats (250–300 g) were stunned and then decapitated. The brain was removed rapidly, the cerebrocortex detached from its internal structures, placed in Tris HCl 50 mmol litre91, pH 7.4 at 4 ⬚C, and homogenized using a tissue tearor (setting 5,5  30-s bursts). 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. Membranes were frozen in aliquots at 940 ⬚C. MEASUREMENT OF

[3H]PN200-110 BINDING

All binding assays were performed in 1-ml volumes of Tris HCl buffer for 90 min (the optimal incubation time was determined empirically) at 20 ⬚C using cerebrocortical membranes (approximately 200 ␮g of protein). Saturation analyses to determine the equilibrium dissociation constant (Kd) and the maximal binding capacity (Bmax) were performed using increasing concentrations of [3H]PN200-110 (0.02–2.0 nmol litre91). In this method, as the concentration of label increases, the binding sites

KAZUYOSHI HIROTA, MD, DAVID G. LAMBERT, PHD, University Department of Anaesthesia, Leicester Royal Infirmary, Leicester LE1 5WW. Accepted for publication: March 22, 1996. Correspondence to D. G. L. †Presented in part at the Anaesthetic Research Society, Manchester Meeting, November 23–24, 1995 (British Journal of Anaesthesia 1996; 76: 591P).

I.v. anaesthetics and L-type calcium channels saturate (analogous to saturation of an enzyme reaction for determination of Km and Vmax) and the concentration at which specific binding saturates is used to determine Bmax (see fig. 1). It follows that the concentration of ligand that yields half the Bmax is the Kd. Non-specific binding was defined in the presence of excess (1095 mol litre91) nifedipine. After incubation, each sample was filtered under vacuum through Whatman GF/B filters using a Brandel cell harvester to separate bound and free radioactivity. Filter retained (bound) radioactivity was extracted for at least 8 h in 4 ml of scintillation fluid before estimation using a beta scintillation counter. The estimated radioactivity was quench corrected with an average [3H] counting efficiency of 60.4 %. In order to determine if an agent interacted with L-type Ca2; channels, the dihydropyridine binding site was labelled with [3H]PN200-110. Increasing concentrations of unlabelled displacer (anaesthetic agents) were then added. Displacement of [3H]PN200-110 binding by the anaesthetic agents indicates that they are interacting at the same site as the radiolabel, that is the ␣1 subunit of the L-type Ca2; channel. A fixed concentration of [3H]PN200-110 (0.2 nmol litre91) was displaced by the following unlabelled agents: thiopentone (n : 6; 1097–1093 mol litre91), pentobarbitone (n : 6; 1097–1093 mol litre91), ketamine (n : 6; 1094–1092 mol litre91, n : 4; 1097–1093 mol litre91), etomidate (n : 6; 3  1097–1094 mol litre91), propofol (n : 6; 3  1097–1094 mol litre91) and alphaxalone (n : 6; 3  1097– 1094 mol litre91). A non-anaesthetic barbiturate, barbituric acid (n : 6; 1097–1093 mol litre91) and nifedipine (n : 5; 1095–3  10913 mol litre91) were examined as negative and positive controls, respectively. Agents were dissolved in Tris HCl buffer as follows; thiopentone (100 mmol litre91 stock in NaOH 0.1 mol litre91), pentobarbitone and barbituric acid (50 mmol litre91 stock in distilled water), propofol (100 mmol litre91 stock in DMSO), alphaxalone and nifedipine (50 mmol litre91 stock in DMSO), etomidate (50 mmol litre91 stock in HCl 0.1 mol litre91) and ketamine (500 mmol litre91 stock in distilled water). Appropriate solvent concentrations were added to control tubes. In addition, a full dose–response curve for the effects of DMSO (n : 6) was constructed and indicated that 1 % DMSO, which is the highest concentration used in these studies, produced only 3 < 1 % displacement. The highest anaesthetic concentration used was limited by agent solubility.

249

Results Binding of [3H]PN200-110 was time- (fig. 1A) and dose- (fig. 1B) dependent. Bmax and Kd values estimated from Scatchard plots (fig. 1C) were 152 (14) fmol/mg protein and 59 (3) pmol litre91, re-

DATA ANALYSIS

Specific binding was calculated as the difference between total and non-specific binding. Bmax and Kd were obtained from Scatchard plots. In displacement studies, the concentrations of displacer producing 25 % and 50 % displacement of specific binding (IC25 and IC50) were obtained by computerassisted curve fitting (Graphpad-Prism) and corrected for the competing mass of [3H]PN200-110 according to Cheng and Prusoff [13] to yield the affinity constant (K25 and K50). All data are expressed as mean (SEM).

Figure 1 Binding of [3H]PN200-110 to rat cerebrocortical membranes was time- (A) and dose- (B) dependent. In A, [3H]PN200-110 concentration was approximately 0.2 nmol litre91. C : Scatchard transformation of the specific binding curve in B (● : specific, ▲ : total and 䉭 : nonspecific binding) and was used to determine Bmax and Kd. Data are mean, SEM (n : 5) in A and from a representative experiment (from n : 5) in B and C.

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British Journal of Anaesthesia Table 1 Effects of anaesthetics on [3H]PN200-110 binding (mean (SEM)) Agent

n

K50 (␮mol litre91)

K25 (␮mol litre91)

Thiopentone Pentobarbitone Propofol Etomidate Alphaxalone Ketamine

6 6 6 6 6 6

134 (6) 211 (6) 97 (5) 36 (1) 34 (5) 377 (49)

48 (2) 95 (7) 40 (2) 25 (2) 17 (3) 198 (16)

Figure 3 Correlation between binding K25, peak serum concentrations and wakening plasma concentrations of alphaxalone (AP), etomidate (ED), propofol (PF), thiopentone (TP) and pentobarbitone (PB). ● : Awake (r 2 : 0.0880, P : 0.0184), ! : peak (r2 : 0.824, P : 0.0333).

with K25 and K50 values shown in table 1. Barbituric acid was ineffective. Ketamine displaced [3H]PN200110 only at high, local anaesthetic concentrations [14, 15] (fig. 2C). With the exception of ketamine, there was a significant correlation between K25 (r2 : 0.834, P : 0.0333) (fig. 3) and K50 (r2 : 0.960, P : 0.0035) and peak plasma concentrations during anaesthesia [16–18]. In addition, there was a significant correlation between plasma concentrations on wakening and both K25 (r2 : 0.843, P : 0.0278) and K50 (r2 : 0.965, P : 0.0028) (fig. 3).

Discussion

Figure 2 [3H]PN200-110 binding (approximately 0.2 nmol litre91) was displaced dose-dependently by anaesthetic barbiturates (thiopentone (!), pentobarbitone (●), barbituric acid (䉭)) (A), propofol (!), etomidate (●), alphaxalone (䉭) and ketamine (▲) at anaesthetic concentrations (B) and ketamine (!) at local anaesthetic concentrations (C). Nifedipine (▼) is included as a high affinity reference compound. Data are mean, SEM (n : 4–6).

spectively. Nifedipine displaced [3H]PN200-110 binding with a K50 of 3.2 (0.6) nmol litre91 (n : 5). All i.v. anaesthetics displaced specific [3H]PN200110 binding in a dose-dependent manner (fig. 2A–C)

In this study, our data suggest that i.v. anaesthetic agents interact with the DHP binding site on neuronal L-channels based on the observed correlation between K25 and K50 and peak plasma concentrations during major surgery or deep sedation [16–18], and plasma concentrations seen on awakening from anaesthesia [17, 19, 20]. Recent reports, however, have suggested that the GABAA receptor may represent a critical target site for general anaesthesia with voltage-sensitive Ca2; channels being less or unimportant as several anaesthetics produce stereospecific inhibition of Cl9 flux through the GABAA receptor and the IC50 for Ca2; channel inhibition by volatile anaesthetic agents exceeds MAC[19]. One important question for consideration is what degree of inhibition is clinically relevant?

I.v. anaesthetics and L-type calcium channels Should a comparison with 50 % inhibition of in vitro events be made? It is inconceivable that the central nervous system or cardiovascular system could withstand this degree of inhibition, yet this comparison has been made (e.g. MAC in the whole animal compared with IC50 channel activity [19]). Several studies [19, 21, 22] have suggested that clinically relevant concentrations of anaesthetics inhibit neurotransmitter release with only 20–30 % inhibition in Ca2; entry. We have therefore argued that it may be more relevant to compare lower degrees of inhibition, for example 25 % [23]. The data in this article used total peak serum concentrations and those seen on wakening [16–20]. After accounting for varying degrees of protein binding the free drug concentration would be lower. It has been suggested that the relevant anaesthetic concentration for in vitro studies is the free (unbound) aqueous concentration at the appropriate end-point for general anaesthesia. However, it should be borne in mind that many i.v. anaesthetic agents [24–27] are concentrated in the brain and their concentration at the target site (the channel) may be similar to the concentration in the brain. As only unbound anaesthetics can pass through the blood–brain barrier, the degree of protein binding is clearly important. However, we should also consider that a new equilibrium which restores the free concentration occurs after a proportion of the free anaesthetic has left the plasma and entered the brain. Therefore, both free and bound (as a pool to replenish the free drug) anaesthetics could be available for diffusion, although the diffusion rate must be dependent on free concentration [28]. In our study, displacement of [3H]PN200-110 binding with i.v. anaesthetics was measured at 90 min (a time at which equilibrium with the brain would have occurred), and therefore we feel that “total” drug concentration may reflect that seen at the target site in the brain. Interestingly, Winters and colleagues [29] examined the influence of albumin on thiopentone action in the rate. In their study they showed that although albumin 400 mg kg91 i.v. 15 min before thiopentone reduced the sleeping time by 30 % (P  0.01), the awake concentration of thiopentone in the brain and total plasma concentration was not affected. If only the free concentration is important, a higher plasma concentration of thiopentone at awakening would be expected in the albumin group. Although their study supports our hypothesis, detailed studies are required to estimate accurately the true “at the target” concentration. In this study we have compared the K50 obtained using rat brain with human serum concentrations during anaesthesia. Although anaesthetic requirements differ between species, there is still close correlation for anaesthetic potencies between species (e.g. a good correlation exists between human MAC and mouse ED50 to abolish the righting reflex [30]). Therefore, we suggest that the K50 obtained in this study using rat cerebrocortical membranes is likely to be comparable with human serum concentrations. Several in vivo studies [3–5] have suggested that the neuronal L-type Ca2; channel DHP binding site may be involved in the mechanism of general

251 anaesthesia. Dolin and co-workers found that the DHP L-type Ca2; channel blocker, nitrendipine, increased the general anaesthetic potencies of ethanol and pentobarbitone [3], and benzodiazepines [4], while the DHP L-channel activator, Bay K 8644, reduced the anaesthetic action of benzodiazepines [4]. Horvàth, Szikszay and Benedek [5] also reported that Bay K 8644 significantly increased the onset time of hypnosis and decreased the anaesthetic potency of dexmedetomidine, while verapamil (Lchannel blocker) significantly augmented the hypnotic–anaesthetic action. However, if L-channels are target sites for anaesthetic agents then why are Lchannel blockers not anaesthetic agents in their own right? This may be explained by poor access to the brain for some L-channel blockers [31]. Interestingly, in a long-term, multicentre study, flunarizine, a non-selective Ca2; channel antagonist which does pass the blood–brain barrier, was reported to produce somnolence as a side effect in 42 % of migraine patients [32]. In addition, Lchannel blockers have been reported to exert an antinociceptive action, which is an important component of general anaesthesia, for both rats and humans [33–36]. Electrophysiological studies also showed that pentobarbitone [9] and propofol [10] inhibited neuronal L-type Ca2; channels. Moreover, volatile anaesthetics have been reported to inhibit the DHP binding site in rat brain membranes [11, 12] consistent with our hypothesis. Collectively we feel that these data suggest that the DHP binding site on the L-type voltage sensitive Ca2; channel may be a target site for general anaesthetic agents. Ketamine could only inhibit DHP binding at concentrations in excess of those required to produce general anaesthesia. However, ketamine has local anaesthetic properties because it can produce not only general anaesthesia but also local anaesthesia (such as i.v. regional anaesthesia) [14, 37]. Durrani and colleagues [14] reported that  0.3 % ketamine (approximately 1092 mol litre91) produced adequate regional anaesthesia with complete sympathetic, sensory and motor block. These concentrations are in agreement with our studies and suggest that the local anaesthetic action of ketamine may include Ca2; channel block. Clearly a wide range of local anaesthetic agents will need to be examined to confirm or refute this hypothesis, although recent studies suggest that Ca2; channels may be involved in the mechanism of action of local anaesthetics [38, 39]. It should also be noted that ketamine is an exception to the “GABA” hypothesis in that this agent does not interact with GABAA receptors [19]. As ketamine is a non-competitive NMDA channel blocker [40] it is possible that this agent could similarly block the aqueous pore of the L-channel. Indeed, channel block by ketamine is supported by the observed inhibition of K;-evoked noradrenaline release from SH-SY5Y human neuroblastoma cells [41] with simultaneous inhibition of K;-evoked Ca2; entry. Does inhibition of DHP binding correlate with inhibition of Ca2; influx? Certainly, Toll [42] showed, using PC12 cells, that the IC50 of nifedipine (2.7 (0.3) nmol litre91) for 45Ca2; uptake was similar to that (1.6 (0.4) nmol litre91) of [3H]nitrendipine

252 binding. Other investigators have also suggested that nifedipine reduces Ca2; influx, with IC50 values using neuronal cells such as NCB-20 cells [43], NG08-15 cells [44] and PC12 cells [21] similar to the K50 reported here. However, while we suspect that DHP displacement with anaesthetic agents may produce a similar reduction in Ca2; influx we have no data in this tissue to support this notion. However, in SH-SY5Y human neuroblastoma cells we have shown [45] that thiopentone and propofol inhibited K;-evoked increases in intracellular Ca2; with IC50 values of 121 and 124 ␮mol litre91, respectively, in good agreement with the K50 values also reported here. In conclusion, we suggest that the DHP binding site of L-type voltage sensitive Ca2; channels may be a target site for i.v. anaesthetic agents.

Acknowledgements We thank Zeneca Pharmaceuticals, Rhone-Poulenc Rorer and Janssen Pharmaceuticals for provision of pure propofol, thiopentone and etomidate, respectively.

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