The modulation of neuron-to-neuron signalling, especially synaptic transmission via the ligand-gated ion channel, plays an important role in general anaesthesia. Volatile anaesthetics at clinically relevant concentrations affect the signal transmission through ligand-gated ion channels [1,2] and the modulators of these channels change the minimum alveolar concentration (MAC) of these anaesthetics . The P2X purinoceptor, i.e. the extracellular adenosine triphosphate (ATP)-gated cation channel, is a member of these ligand-gated ion channels. This receptor is responsible for a fast excitatory neurotransmission in the central nervous system (CNS) . Several recent studies on the purinoceptor indicate various functional roles including the conduction of nociceptive stimulus [5,6]. The intracerebroventricular administration of P2 antagonists reduces the MAC of volatile anaesthetics , which suppress the ATP-induced inward current in neurons of the locus coeruleus , thus suggesting that the purinergic receptor is one of the targets of the volatile anaesthetics. In general, the spinal cord is the principal sites of action for volatile anaesthetics with respect to MAC measurements . However, the descending modulatory system influences the action of anaesthetics on the cord . The neurons of the locus coeruleus also project norepinephric axons widely to many regions of the brain including the cortex, hippocampus and thalamus - all of which are important targets for general anaesthesia . Indeed, the destruction of the locus coeruleus results in reduction of MAC . It is therefore possible that the suppression of P2X receptor activity by P2 antagonists or volatile anaesthetics in the brain could be involved in anaesthetic effects.
The study was undertaken to investigate the mechanism of the suppressive actions of volatile anaesthetics on P2X receptors. The hypothesis was that inhibition of neurotransmission may occur if volatile anaesthetics block the binding of ATP to the P2X receptor. ATP not bound to its receptor is readily hydrolysed to adenosine without activation of the P2X receptor. Adenosine is known to be an inhibitory neurotransmitter . Therefore, the effects of volatile anaesthetics on [3H] α,β-methylene ATP, a selective P2X agonist, binding in rat crude synaptic membranes were investigated.
This study was approved by the Animal Research Committee at Jikei University School of Medicine.
Preparation of rat crude synaptic membranes
The crude synaptic membranes were essentially prepared according to the method of Zukin and colleagues . Male Sprague-Dawley rats (7 weeks old, 150-200 g) were anaesthetized with thiopental 50 mg kg−1 intraperitoneally and the brain rapidly excised. Thiopental does not to change the P2X receptor activity in PC12 cells . The brain was homogenized with sucrose solution (0.32 molar, 9 vols) and centrifuged at 1000g for 10 min. The pellet was dispersed and disrupted in Tris-HCl buffer (10 mmol, 20 vols, pH 7.4), and centrifuged at 8000g for 15 min. The supernatant and soft layer were resuspended with the buffer and stored overnight at −20°C. The suspension was thawed and centrifuged at 25 000g for 15 min. The resultant pellet was washed five times with 20 times the volume of the buffer and stored at −20°C until use.
Assay of [3H] α,β-methylene ATP binding
The incubation was initiated by addition of [3H] α,β-methylene ATP (10 nmol) for 15 min at 4°C with Tris 10 mmol, 100 μg crude synaptic membranes protein, and the volatile anaesthetics (sevoflurane or isoflurane), or the P2 antagonists (suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS)), to a total volume 200 μL. The binding assay was performed at 4°C because the specific [3H] α,β-methylene ATP binding in mouse crude synaptic membranes is higher at 4°C than at 25°C . The MACs of sevoflurane and isoflurane in the Sprague-Dawley rat were 2.40 and 1.46%, respectively [17,18]. The corresponding aqueous concentrations (EC50) were 0.40 and 0.35 mmol at 37°C for sevoflurane and isoflurane, respectively . EC50 concentrations for general anaesthesia decreased with temperature in aqueous concentrations. Applying the equation calculated by Franks and Lieb , the estimated EC50 for sevoflurane and isoflurane were 0.24 and 0.22 mmol at 4°C, respectively. Therefore, the concentrations up to 1.5 mmol were selected to examine the effect of volatile anaesthetics at clinically relevant concentrations. The reaction was terminated by centrifugation (18 000g for 10 min), and the pellet was rinsed rapidly and superficially with ice-cold deionized water. The radioactivity in the pellet was measured with a liquid scintillation counter. Specific [3H] α,β-methylene ATP binding was determined by subtracting non-specific binding (the amount of radioactivity in the pellet in the presence of unlabeled ATP 1 mmol) from total binding (the amount of the radioactivity in the absence of unlabeled ATP). At each concentration of volatile anaesthetics and P2 antagonists, the data were obtained from four separate experiments in which the determinations were carried out in triplicate.
A parametric test was used. Data were analysed by analysis of variance (ANOVA) with subsequent intragroup comparisons using Scheffé's F-test. P < 0.05 was considered as statistically significant.
Figure 1 shows that sevoflurane and isoflurane did not affect [3H] α,β-methylene ATP binding at clinically relevant concentrations up to 1.4 and 1.5 mmol, respectively. Suramin, one of the P2 antagonists, decreased [3H] α,β-methylene ATP binding (68.7 ± 14.7% at 10 μmol, 49.5 ± 6.4% at 50 μmol, 24.3 ± 5.7% at 100 μmol, n = 12, means ± SD) in a dose-dependent manner. The decrease of the binding by suramin was significant at 10, 50 and 100 μmol. However, PPADS, another P2 antagonist, did not affect the binding up to 100 μmol; this is similar to the effect of the volatile anaesthetics (Fig. 2). Scatchard analyses were performed with [3H] α,β-methylene ATP (0.5-50 nmol) and suramin (50 μmol), and revealed that suramin blocked [3H] α,β-methylene ATP binding non-competitively. Bmax and Kd were as follows: Bmax, control: 3.43 ± 0.45, suramin; 1.62 ± 0.35 pmol mg−1 protein; Kd, control: 21.2 ± 3.4, suramin; 20.7 ± 2.7 nmol (n = 12, mean ± SD). There were no significant differences in Scatchard analysis between control, volatile anaesthetics and PPADS.
The major findings were that sevoflurane and isoflurane did not affect the binding of the ATP analogue, [3H] α,β-methylene ATP, to the P2X receptor. It has been demonstrated that intracerebroventricular administration of suramin and PPADS reduced the MAC of volatile anaesthetics, suggesting that the inhibition of the ATP signalling system in the CNS leads to anaesthetic and analgesic effects . Therefore, it is possible that this system could be a target for the action of the volatile anaesthetics. However, in contrast to our hypothesis, the blockade of ATP binding to the P2X receptor cannot be the site or mechanism of action of the volatile anaesthetics. It was also reported that ATP evoked a rapidly rising and moderately desensitizing inward current in neurons of the locus coeruleus; also, that sevoflurane at clinically relevant concentrations significantly suppressed this current in a dose-dependent manner . The ATP-induced current was likely to be mediated by P2X receptors, and the current sensitive to sevoflurane was blocked by PPADS, indicating that the attenuation of extracellular ATP-mediated signalling in the CNS is one of the multiple actions of volatile anaesthetics. Taking the results of the present study into account, it can now be suggested that volatile anaesthetics could attenuate the signalling, via P2X receptors, by modulation of receptor function after the binding of ATP to the P2X receptor because (a) intracerebroventricular administration of P2 antagonists reduced the MAC of volatile anaesthetics, (b) ATP-induced currents were inhibited by volatile anaesthetics and (c) volatile anaesthetics did not affect [3H] α,β-methylene ATP binding.
The binding of [3H] α,β-methylene ATP was significantly inhibited by suramin, but not by PPADS. The two P2 antagonists showed differential effects on the binding activity of [3H] α,β-methylene ATP in the current study. These differential effects of P2 antagonists on the binding in P2X receptors suggest that the antinociceptive effect of suramin stems, at least in part, from the inhibitory effect on the binding. On the other hand, PPADS might reduce the MAC of volatile anaesthetics in a similar way to volatile anaesthetics, interfering with the receptor function itself. The exact mechanism for the differential effect between suramin and PPADS is not apparent; however, these structurally different compounds have several different properties. The sensitivity of both antagonists for cloned P2X receptor sub-types is not same . It has been demonstrated that suramin  and the suramin analogue  are very sensitive to the heteromeric P2X receptors. The differential sensitivity of both antagonists for P2X receptor sub-types might be due to the differential binding activity for P2X receptor. Suramin may bind more preferably the P2X receptors - which are rich in the crude synaptic membranes used in this study - than PPADS. Beside P2X receptors, suramin might slow the onset of glutamate-evoked AMPA current, while PPADS did not show any effect on this current . N-methyl-D-aspartate (NMDA) and glycine-induced inward currents were also significantly blocked by suramin, but not by PPADS . The present study reports another differential effect between suramin and PPADS: that suramin but not PPADS decreased the binding of [3H] α,β-methylene ATP in rat crude synaptic membranes.
Like the P2X receptor, volatile anaesthetics did not affect the agonist binding at the NMDA  and nicotinic acetylcholine (nACh) receptors . Martin and colleagues demonstrated that enflurane inhibited glutamate-stimulated [3H] MK-801, but not [3H] CGS-19775, binding to the NMDA receptor channel . MK-801 and CGS-19775 are the NMDA receptor ligands that bind to the sites within the channel and glutamate binding sites, respectively. These data suggest that enflurane does not affect glutamate binding sites in the NMDA receptor but rather interferes with conformational changes of the receptor necessary for channel activation. These finding are consistent with our results in terms of the anaesthetic interaction with receptor functions but not the agonist binding. In the nACh receptor, the binding domain of volatile anaesthetics on the ligand-gated ion channel receptor was determined. [14C] halothane photoaffinity labelling of both the native Torpedo membrane and the isolated nACh receptor was reduced by unlabelled halothane but not by the agonist and antagonist of nACh receptors, suggesting that the agonist site is not a likely candidate for halothane binding. The fact that volatile anaesthetics did not bind to the agonist binding sites is in agreement with our findings whereby volatile anaesthetics did not influence the binding of agonist to a P2X receptor.
In conclusion, the suppressive effect of volatile anaesthetics on the ATP signalling system might be one of the multiple actions of these anaesthetics. However, the blockade of ATP binding to the P2X receptor is not the mechanism of action of volatile anaesthetics.
Work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan, and the Japan Health Science Foundation. The authors thank Dr Salim Hayek (Cleveland Clinic Foundation, Cleveland, OH, USA) for help with manuscript preparation.
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