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British Journal of Anaesthesia 1995; 75: 326–329

Anaesthetic agents decrease the activity of nitric oxide synthase from human polymorphonuclear leucocytes H. F. GALLEY, L. R. NELSON AND N. R. WEBSTER

Summary Nitric oxide (NO) inhibitors reduce the threshold for anaesthesia. We have investigated the action of anaesthetic agents on human nitric oxide synthase (NOS) activity. Thiopentone reduced mean NOS activity to 36.6 (SD 8.9) % of control at 100 ␮mol litre⫺1 (P ⬍ 0.001) and 50.9 (20.3) % at 1 mmol litre⫺1 (P ⬍ 0.05). Ketamine showed similar effects, with activity reduced to 67.0 (17.6) % (P ⬍ 0.05) and 57.7 (8.5) % (P ⬍ 0.001) at 100 ␮mol litre⫺1 respectively. Etomidate and 1 mmol litre⫺1, 100 ␮mol litre⫺1 did not significantly alter activity (88.2 (8.1) %) but 1 mmol litre⫺1 did (60.6 (10.4) %, P ⬍ 0.005). Halothane also caused a significant decrease in NOS activity at all concentrations. This effect was specific as other enzymes were unaffected. We conclude that anaesthetic agents have a profound effect on NOS activity and as inhibition of NO release augments anaesthesia, we suggest that this may play a role in the mechanism of anaesthesia in humans. (Br. J. Anaesth. 1995; 75: 326–329). Key words Anaesthetics i.v., thiopentone. Anaesthetics i.v., etomidate. Anaesthetics i.v., ketamine. Anaesthetics volatile, halothane. Enzymes, nitric oxide synthase. Blood, leucocytes.

Nitric oxide (NO) is a potent vasodilator produced from L-arginine in a variety of cells, including vascular endothelium, in response to physical and receptor stimulation, by the action of a constitutive calcium–calmodulin–dependent enzyme, nitric oxide synthase (NOS). NO is necessary for the physiological regulation of vasodilator tone, causing activation of soluble guanylate cyclase in vascular smooth muscle cells, release of cyclic GMP and thus vascular relaxation. NO has several functions in addition to its vasodilator action [1]. NOS catalyses the five-electron reduction of L-arginine to citrulline and NO, and demonstrates significant amino acid sequence homology to NADPH–cytochrome P450 reductase [2]. Enflurane, isoflurane and halothane have been shown to decrease receptor generated NO-induced vascular relaxation [3–8] but not that induced by exogenous NO-generating agents such as sodium nitroprusside [3, 5, 6], although the exact mechanisms remain unclear. There are no reports of the influence of either volatile or i.v. anaesthetic agents on the activity of human NOS. We have shown

previously NOS activity in human polymorphonuclear leucocytes (PMN) [9] and have used this ex vivo model to investigate the action of anaesthetic agents on NOS activity in humans.

Methods PREPARATION OF CELL LYSATES

Heparinized venous blood was obtained on several occasions from a single healthy volunteer for all experiments. Polymorphonuclear leucocytes (PMN) were separated using a single step commercially available density gradient, Polymorphprep (Nycomed UK, Birmingham, UK), as described previously [9]. Briefly, 10 ml of whole blood was layered onto 2
3.5 ml of Polymorphprep, centrifuged and the mononuclear cells discarded. PMN were removed, washed in RPMI 1640 medium without glutamine (ICN Biomedicals Ltd, Thame, Oxfordshire, UK) containing HEPES buffer 20 mmol litre⫺1 (ICN), and any contaminating erythrocytes removed by hypotonic shock. Cells were then washed twice in phosphate-buffered saline (PBS, Dulbecco’s A, pH 7.3, Oxoid-Unipath Ltd, Basingstoke, Hampshire, UK) and lysed in 1 ml of double-distilled water. The lysate was sonicated for 1 min and endogenous arginine was removed by incubating with :100 mg of washed DowexAG50W-X8 resin (Bio-Rad Laboratories Ltd, Hemel Hempstead, Hertfordshire, UK) for 5 min. After centrifugation at 10 000 g, the clear supernatant was stored at ⫺70 °C for NOS assay. NITRIC OXIDE SYNTHASE ACTIVITY ASSAY—I.V. ANAESTHETIC AGENTS

The assay for NOS is based on the quantitative conversion of oxyhaemoglobin to methaemoglobin by NO, which can be followed spectrophotometrically as a decrease in absorbance at 413 nm (fig. 1). The activity of NOS can be calculated from the reaction rate using the molar extinction coefficient for methaemoglobin, which we have found previously to be 37.2 (mmol litre)⫺1 cm⫺1 [9]. The HELEN F. GALLEY*, FIMLS PHD, LISA R. NELSON, HNC, NIGEL R. WEBSTER*, BSC, MB, CHB, PHD, FRCA. Clinical Oxidant Research Group, St James’s University Hospital, Leeds, UK. Accepted for publication: April 3, 1995. *Present address: University of Aberdeen, Anaesthesia and Intensive Care, Foresterhill, Aberdeen AB9 2ZD. Correspondence to H.F.G.

Anaesthetic agents decrease NOS activity

327 SUPEROXIDE DISMUTASE ASSAY

The effect of anaesthetic agents on superoxide dismutase (SOD) activity was investigated using a commercially available spectrophotometric kit (R&D Systems Europe, Oxford, UK). The activity of bovine SOD 500 u. ml⫺1 was measured in the presence of etomidate, thiopentone and ketamine at final concentrations of 100 ␮mol litre⫺1, 1 mmol litre⫺1 and 10 mmol litre⫺1, and compared with activity without the test agents. Bovine SOD was exposed also to 0–4 % halothane before assay, as described for NOS. In addition we investigated the direct effect of the anaesthetics on the assay system in the absence of SOD. Figure 1 Reaction of nitric oxide with oxyhaemoglobin (OxyHb) to form methaemoglobin (Met-Hb) results in a decrease in absorbance at 413 nm which is proportional to the amount of nitric oxide present.

technique used in the present study is the same as that described previously [9] with some modifications. An incubation mixture containing superoxide dismutase 100 u. ml⫺1 (from bovine erythrocytes, Sigma-Aldrich Ltd, Poole, Dorset, UK), catalase 2000 u. ml⫺1 (from bovine liver, Sigma) and human oxyhaemoglobin 1.29 ␮mol litre⫺1, prepared as described previously [9] in PBS, was pre-warmed to 37 °C. Into 1-cm cuvettes were placed 0.5 ml of the incubation mixture, 0.2 ml of cell lysate and 0.1 ml of etomidate (Hyponomidate, Janssen Pharmaceuticals, Wantage, Oxfordshire, UK), ketamine hydrochloride (Ketalar, Parke-Davis, Pontypool, Gwent, UK) or thiopentone (Intraval Sodium, May and Baker Ltd, Dagenham, Kent, UK) at concentrations of 0, 100 ␮mol litre⫺1 and 1 mmol litre⫺1. The cuvettes were then placed in a spectrophotometer (Lambda 2, with PECSS software package, Perkin Elmer, Beaconsfield, Bucks, UK) maintained at 37 °C, and the reaction was initiated by the addition of 0.1 ml of ␤ NADPH 2 ␮mol litre⫺1 (Sigma) and 0.1 ml of L-arginine 0.3 mol litre⫺1 (Sigma). Absorbance was recorded at 413 nm for 3 min and the slope found, to give a reaction rate (change in absorbance units per minute). NITRIC OXIDE SYNTHASE ACTIVITY ASSAY —HALOTHANE

After washing in PBS, isolated cells were resuspended in PBS and 0.2-ml aliquots placed in small uncapped conical tubes. These were then positioned inside a 30-ml capacity gas-tight glass vessel, into which had been pipetted a volume of halothane (Fluothane, ICI Pharmaceuticals, Macclesfield, Cheshire, UK), calculated to give final concentrations of 0, 1, 2, 3 or 4 % in air. The vessels were capped and allowed to equilibrate. Cells were exposed to halothane for 30 min and then centrifuged, lysed and sonicated as previously. For the NOS assay, cell lysates were treated as described above, except that 0.1 ml of PBS was added instead of the i.v. anaesthetic agents. The direct effect of the anaesthetic agents on the assay system was also investigated in the absence of NOS.

L-ARGININE MEASUREMENT

The effect of the anaesthetic agents on the stability of L-arginine was assessed by high performance liquid chromatography (HPLC) [10]. L-Arginine 30 mmol litre⫺1 in PBS was incubated with 100 ␮l of etomidate, thiopentone or ketamine 10 mmol litre⫺1 for 30 min, and then diluted in water. Arginine was derivatized with 9-fluorenylmethyl chloroformate 15 mmol litre⫺1 (Sigma) in acetone and nonderivatized material was removed by pentane (Sigma) extraction. The supernatant fraction (20 ␮l) was then injected onto a Hypersil C18 3 ␮m reverse phase column (Jones Chromatography, Hengoed, Mid-Glamorgan, UK), and separated by gradient elution. The eluent was varied linearly from acetonitrile–methanol–acetic acid buffer, pH 4.2 (10:40:50), to acetonitrile–acetic acid buffer (50:50) over 9 min, and arginine was detected fluorimetrically at an excitation wavelength of 260 nm and an emission wavelength of 313 nm. STATISTICAL METHODS

Statistical analysis was performed using the computer program Fig P (Biosoft, Cambridge, UK). Results are expressed as mean (SD) percentages of the activity in the absence of anaesthetic, and represent five replicate experiments. Synthase activity in cell lysates after exposure to each concentration of anaesthetic agent was compared with activity in the absence of anaesthetic by Student’s paired t test, using the raw data, that is before conversion to percentage activity for presentation purposes. Differences were taken to be significant when P  0.05.

Results NITRIC OXIDE SYNTHASE

NOS activity isolated from PMN was reduced in the presence of all anaesthetic agents tested. Thiopentone reduced the activity to 36.6 (8.9) % of control activity at 100 ␮mol litre⫺1 (P ⬍ 0.001) and 50.9 (20.3) % at 1 mmol litre⫺1 (P ⬍ 0.05, fig. 2A). Ketamine showed a similar effect, with activity reduced to 67.0 (17.6) % (P ⬍ 0.05) and 57.7 (8.5) % (P ⬍ 0.001) at 100 ␮mol litre⫺1 and 1 mmol litre⫺1, respectively (fig. 2B). With etomidate 100 ␮mol litre⫺1, the activity of NOS was not significantly

328

British Journal of Anasthesia

Figure 3 Effect of 1–4 % halothane exposure of human polymorphonuclear leucocytes on nitric oxide synthase (NOS) activity. Results are expressed as percentage of the activity in the absence of halothane (control) ( ) and are the mean (SD) of five replicate experiments. Significantly lower (**P ⬍ 0.01) compared with control.

L-ARGININE

We were unable to show any reduction in concentration when arginine was exposed to anaesthetic agents.

Discussion

Figure 2 Effect of thiopentone (A), ketamine (B) and etomidate (C) 100 ␮mol litre⫺1 and 1 mmol litre⫺1 on nitric oxide synthase (NOS) activity from human polymorphonuclear leucocytes. Results are expressed as percentage of the activity in the absence of anaesthetic (control) ( ) and are the mean (SD) of five replicate experiments. Significantly lower activity (*P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001) compared with controls.

altered (88.2 (8.1) %) but activity was reduced at 1 mmol litre⫺1 (60.6 (10.4) %, P ⬍ 0.005) (fig. 2C), Halothane also caused a significant decrease in NOS activity at all concentrations (fig. 3). The anaesthetic agents alone had no effect on the assay system for NOS. SUPEROXIDE DISMUTASE

None of the anaesthetic agents had any effect on SOD activity at any concentration studied, and no agent interfered with the measurement of SOD by the assay used.

We have demonstrated a profound effect of both volatile and i.v. anaesthetic agents on NOS activity from isolated human PMN. Previous animal studies have demonstrated inhibition of receptor generated NO-dependent relaxation of aortic rings by volatile anaesthetic agents [3–8]. Many explanations have been suggested, including interaction with cyclic GMP phosphodiesterase enzymes [4], modification of the half-life of NO [8], interference with the release of NO [3] or stimulation of the release of a constricting factor [7]. The inhalation anaesthetics, halothane and isofluorane, have been shown to reduce rat brain NOS activity, but in contrast with the present study, these workers found no effect of pentobarbitone, ketamine, fentanyl or midazolam [11]. Variation in NOS from different cell types is apparent [12]. Cytosolic NOS isolated from rat PMN is calcium-dependent, but calmodulin-independent [13], while that from rat brain is dependent on both calcium and calmodulin [14]. The isoform induced by endotoxin–cytokine exposure in murine macrophages is dependent on neither [12]. In rat PMN, 70 % of the enzyme activity is cytosolic, and 30 % is particulate, with markedly different specific activities, whereas that from rat brain is entirely soluble [13]. There have been no similar studies in human cell types, and it is not known if significant species variation occurs. This is the first study of the effect of anaesthetic agents on NOS activity in human cells. SOD was included in the assay for NOS in the present study to remove any superoxide, as the presence of superoxide would reduce apparent NOS activity by reacting preferentially with NO [9, 15]. The effect of the anaesthetic agents on the activity of

Anaesthetic agents decrease NOS activity SOD was investigated therefore in order to preclude inadequate removal of superoxide as an explanation for apparent reductions in NOS activity. The anaesthetic agents used did not have any effect on SOD activity or any inhibitory effect on the assays used to measure either SOD or NOS. The mechanisms of the inhibitory action of the anaesthetics on NOS activity are speculative. The effect of i.v. and volatile anaesthetics on NOS may work via different mechanisms. In the present study, whole cells were exposed to halothane, rather than cell lysates as for thiopentone, etomidate and ketamine. Halothane has been shown to inactivate NADPH–cytochrome P450 reductase, an enzyme which has significant amino acid sequence homology to NOS [2]. Our studies of the lack of effect of anaesthetics on SOD, and other enzyme studies [16], show that this enzyme inhibition is clearly not a nonspecific phenomenon. It is possible that the anaesthetic agents or their metabolites may interfere in some way with the availability of L-arginine. However, investigation of the interaction of ketamine, etomidate and thiopentone with L-arginine using HPLC failed to find any evidence to support this explanation. It has been shown in animal studies that the anaesthetic effect of both i.v. and volatile anaesthetic agents is augmented by NO inhibitors, such that the threshold for anaesthesia is reduced [17, 18]. NO is thought to function as a neurotransmitter involved in excitory amino acid-mediated neurotoxicity, consciousness, learning and memory [19]. Cellular influxes of calcium are promoted by increased concentrations of excitatory amino acids, through activation of N-methyl-D-aspartate (NMDA) receptors. In rats, antagonists of NMDA receptors reduce the minimal alveolar concentration for isofluorane [20] and NO inhibitors block spinal sensitization mediated by NMDA receptors [21]. Ketamine in particular is a known NMDA antagonist, and pentobarbitone enhances GABAmediated inhibitory neurotransmission, thus indirectly increasing excitatory amino acid neurotransmission. Data from these studies and the present study strongly suggest that NO plays a role in the molecular mechanism of anaesthesia in humans, possibly via interaction with NMDA receptors, in both i.v. and inhalation anaesthesia.

Acknowledgements The work was performed in the laboratories of the Clinical Oxidant Research Group, St James’s Hospital, Leeds, UK. Financial support was received from the Medical Research Council, the Sir Jules Thorn Charitable Trust and Yorkshire Health.

References 1. Moncada S, Higgs EA. Biological relevance of the Larginine:nitric oxide pathway. In: Moncada S, Nistico G, Higgs EA, eds. Nitric Oxide: Brain and Immune System. London: Portland Press Proceedings, 1993; 1–12.

329 2. White KA, Marletta MA. Nitric oxide synthase is a cytochrome P-450 type hemoprotein. Biochemistry 1992; 31: 6627–6631. 3. Toda H, Nakamura K, Hatano Y, Nishiwada M, Kakuyama M, Mori K. Halothane and isoflurane inhibit endotheliumdependent relaxation elicited by acetylcholine. Anesthesia and Analgesia 1992; 75: 198–203. 4. Eskinder H, Hillard CJ, Flynn N, Bosnjak ZJ, Kampine JP. Role of guanylate cyclase–cGMP systems in halothane induced vasodilation in canine cerebral arteries. Anesthesiology 1992; 77: 482–487. 5. Muldoon SM, Hart JL, Bowen KA, Freas W. Attenuation of endothelium-mediated vasodilation by halothane. Anesthesiology 1988; 68: 31–37. 6. Uggeri MJ, Proctor GJ, Johns RA. Halothane, enflurane and isoflurane attenuate both receptor- and non-receptormediated EDRF production in rat thoracic aorta. Anesthesiology 1992; 76: 1012–1017. 7. Stone DJ, Johns RA. Endothelium-dependent effects of halothane, enflurane and isoflurane on isolated rat aortic vascular rings. Anesthesiology 1989; 71: 126–132. 8. Blaise G, To Q, Parent M, Lagarde B, Asenjo F, Sauve R. Does halothane Interfere with the release, action or stability of endothelium-derived relaxing factor/nitric oxide? Anesthesiology 1994; 80: 417–426. 9. Goode HF, Webster NR, Howdle PD, Walker BE. Nitric oxide production by human peripheral blood polymorphonuclear leucocytes. Clinical Science 1994; 86: 411–415. 10. Einarsson S, Josefsson B, Lagerkvist S. Determination of amino acids with 9-fluorenylmethyl chloroformate and reversed-phase high performance liquid chromatography. Journal of Chromatography 1983; 282: 609–618. 11. Tobin JR, Martin LD, Breslow MJ, Traystman RJ. Selective anesthetic inhibition of brain nitric oxide synthase. Anesthesiology 1993; 79: A693. 12. Förstermann, U, Schmidt HHHW, Pollock JS, Sheng H, Mitchell JA, Warner TD, Nakane M, Murad F. Isoforms of nitric oxide synthase. Characterisation of different cell types. Biochemical Pharmacology 1991; 42: 1849–1857. 13. Yui Y, Hattori R, Kosuga K, Eizawa H, Hiki K, Ohkawa S, Ohnishi K, Terao S, Kawai C. Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils. Journal of Biological Chemistry 1991; 266: 3369–3371. 14. Förstermann U, Gorsky LD, Pollock JS, Schmidt HHHW, Heller M, Murad F. Regional distribution of EDRF/NOsynthesizing enzyme(s) in rat brain. Biochemical and Biophysical Research Communications 1990; 168: 727–732. 15. Gryglewski RJ, Palmer RMJ, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived relaxing factor. Nature (London) 1986; 320: 454–456. 16. Alifimoff JK, Brandom BW, Cook DR. Enflurane, halothane and isoflurane do not inhibit angiotensin converting enzyme. Canadian Anaesthetists Society Journal 1985; 32: 351–357. 17. Johns RA, Moscicki JC, DiFazio CA. Nitric oxide synthase inhibitor dose-dependently and reversibly reduces the threshold for halothane anesthesia. Anesthesiology 1992; 77: 779–784. 18. Bansinath M, Arbabha B, Turndorf H, Garg UC. Hypnotic effect of intravenous anesthetics is augmented by nitric oxide synthase inhibition. Anesthesiology 1993; 79: A694. 19. Hanbauer I. The role of nitric oxide in neurotransmitter release. In; Moncada S, Nistico G, Higgs EA, eds. Nitric Oxide: Brain and Immune System. London: Portland Press Proceedings, 1993; 135–141. 20. Yamamoto T, Shimoyama N, Mizuguchi T. Nitric oxide synthase inhibitor blocks spinal sensitization induced by formalin injection into the rat paw. Anesthesia and Analgesia 1993; 77: 886–890. 21. Kuroda Y, Strebel S, Rafferty C, Bullock R. Neuroprotective doses of N-methyl-D-aspartate receptor antagonists profoundly reduce the minimum alveolar anesthetic concentration (MAC) for isoflurane in rats. Anesthesia and Analgesia 1993; 77: 795–800.