British Journal of Anaesthesia 1994; 73: 820-825

Inhalation anaesthetic competition at high-affinity cocaine bindingsites in rat brain synaptosomes

R. G. ECKENHOFF AND D . FAGAN

Summary

We have shown previously that inhalationanaesthetics inhibit dopamine transport in ratsynaptosomes. In order to determine if this in-hibition is associated with occupancy of thecocaine site, we examined binding of [3H](2p~-carbomethoxy-3p-(4-fluorophenyl)-tropane) (3H-CFT) in the presence of halothane or isoflurane0.01-5 mmol litre"1 in rat brain synaptosomes. Bothanaesthetics inhibited 3H-CFT binding (mean/Ci 0.61 (SEMO.12) and 0.75 (0.21) mmol litre"1,respectively), by increasing /Cd (13.8 (0.6) and 29.8(12.8) nmol litre"1, respectively) compared withcontrol (8.02 (0.5) nmol litre"1) {P < 0.01).Halothane did not change fimax, but isofluraneincreased it significantly. Cocaine protected CFTsites from /V-ethylmaleimide alkylation, but neitheranaesthetic did. Photoaffinity labelling withhalothane significantly inhibited 3H-CFT bindingcompared with UV-exposed controls. We concludethat clinically relevant concentrations of bothanaesthetics inhibit high-affinity CFT binding, andthe data suggest overlapping sites for halothaneand CFT. {Br. J. Anaesth. 1994; 73: 820-825)

Key wordsAnaesthetics volatile. Nerve, synaptosome. Rat.

The inhalation anaesthetics halothane and isofluranehave been shown to non-competitively inhibitdopamine transport into rat brain synaptosomes [1],suggesting allosteric inhibition of the dopaminetransporter. This in vitro result may explain in vivomicrodialysis studies which show an increase inextracellular dopamine concentration with inductionof halothane anaesthesia [2—4]. Furthermore, be-cause systemic administration of the dopamineprecursor, L-dopa, has been shown to reduce theED50 (minimum alveolar concentration, MAC) ofhalothane by approximately 50% in mice [5],elevated synaptic dopamine concentrations may be acomponent of the mechanism of halothane anaes-thesia. Because cocaine and its analogues are alsonon-competitive antagonists of the dopamine trans-porter [6], we hypothesized that the inhalationanaesthetics produce inhibition of the transporter byinteraction at this same site. We explored thisquestion in rat brain synaptosomes and foundevidence for a competitive interaction betweencocaine and halothane, but not isoflurane, which may

explain the previously reported inhibition of thedopamine transporter by these two inhalationanaesthetics.

Materials and methods

CHEMICALS

[3H]2P-Carbomethoxy-3P-(4-fluorophenyl)-tropane(3H-CFT), a potent cocaine analogue [7] with aspecific activity of 83.4 Ci mmol"1, was purchasedfrom DuPont-NEN (Boston, MA, USA). CocaineHC1 was obtained from Astra PharmaceuticalProducts (Westborough, MA, USA), halothane (2-bromo-2-chloro-l,l,l-trifluoroethane) was obtainedfrom Halocarbon Laboratories (Hackensack, NJ,USA) and the preservative thymol was removedusing an activated alumina column [8]. Isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether)was obtained from Anaquest Corp (Murray Hill, NJ,USA). Other chemicals were of analytical grade andwere purchased from Sigma (St Louis, MO, USA).

TISSUE PREPARATION

Animal studies were approved by the InstitutionalAnimal Care and Use Committee. Whole brains(excluding the cerebellum and midbrain, approxi-mately 2 g) of male adult Sprague-Dawley rats wereprocessed as follows: brains were removed rapidlyfrom animals anaesthetized with pentobarbitone50 mg kg"1 i.p., washed, minced and homogenizedin 20 volumes of ice cold sucrose solution0.32 mol litre"1 using a Potter-Elevehjem glasshomogenizer with a Teflon pestle. The crude homo-genate was centrifuged at 1000 # for 15min, thepellet resuspended and homogenized again in sucrose0.32 mol litre"1 and then centrifuged at 1000 £ for15min. The combined supernatants were recen-trifuged at 30000 £ for 30 min. The final pellet(P2 fraction) containing synaptosomal membraneswas resuspended in 5 volumes of 2 °C Tris10 mmol litre^-NaCl buffer 100 mmol litre"1,pH 7.3, and used immediately. Protein concentrationwas determined by Coomassie blue assay [9].Preliminary studies indicated that the low remainingconcentration (< 1 umol litre"1) of barbiturate hadno effect on [3H]CFT binding.

RODERIC G. ECKENHOFF, MD, DANIELLE FAGAN, BA, Departmentsof Anesthesia and Physiology, University of Pennsylvania MedicalCenter, 7 Dulles Bldg, 3400 Spruce Street, Philadelphia, PA19104-4283, USA. Accepted for publication: July 5, 1994.

Correspondence to R. G. E.

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Cocaine binding and inhalation anaesthetics 821

BINDING ASSAYS

Binding experiments were performed in Teflon-sealed microvials (600-ul capacity) containingvarious concentrations of 3H-CFT (see below) inTris 10 mmol litre-'-NaCl buffer 100 mmol litre"1,pH 7.3, and about 300 ug of synaptosomal protein.Included in half of the vials was cocaine100 |imol litre"1 to determine non-specific binding.Vials were incubated at 2 °C for 90-120 min (pre-liminary experiments showed stable binding between30 and 120 min). Bound and free label were sepa-rated by vacuum filtration using Whatman GF/Bniters pre-soaked in 0.05% poly(ethyleneimine).Filters were washed with 9 ml of 2 °C saline154 mmol litre"1 and incubated for at least 12 h withscintillation fluid in vials before counting in an LKBWallac 1209 counter for 3 min.

COMPETITION EXPERIMENTS

The inhalation anaesthetics halothane and isofluranewere added to the microvials as possible competitorsfor 3H-CFT binding. At fixed concentrations of 3H-CFT, increasing concentrations of halothane andisoflurane (0.01 to approximately 5 mmol litre1)were produced by injecting appropriate volumes ofhigher concentrations dissolved in buffer usingHamilton (Reno, NV, USA) gastight syringes intothe capped Teflon-sealed microvials. This finaladdition of anaesthetic solution completely filled themicrovials, which were then vortexed, incubated at2 °C for 2 h, filtered and counted as described above.In order to verify anaesthetic concentrations, parallelmicrovials containing only buffer and experimentalconcentrations of anaesthetic were also incubated for2 h and subjected to gas chromatographic analysisafter hexane extraction. Competition data weretransformed to percent of control (0 mmol litre"1

anaesthetic) and fitted with sigmoid curves withnegative Hill coefficients using non-linear leastsquares regression. Maximum binding (100%) wasconstrained. The inhibition constant (K^ wascalculated from the IC50 using the predetermined Kdfor CFT and the known 3H-CFT concentration inthe experiments.

BINDING ISOTHERMS

In order to determine the anaesthetic influenceon variables of the CFT saturable binding com-ponent, fixed concentrations of anaesthetic (halo-thane 1 mmol litre"1 or isoflurane 1.7 mmol litre"1,final concentration) were produced in the pre-sence of increasing concentrations of 3H-CFT(0.75 nmol litre"1, final concentration). Control ex-periments (0 mmol litre"1 anaesthetic) were also per-formed. Both sets of experiments were conductedat 2 °C with a 2-h incubation period. Scatchardanalysis was used to determine Kd and fimax(EBDA/LIGAND software) [10]. The low CFTconcentration range used did not allow examinationof low-affinity sites reported previously [6, 7], andthus the data were fitted to single site models.

PROTECTION ASSAYS

To assess if the anaesthetics may protect the cocainesite from alkylation, as with cocaine analogues [11],we preincubated synaptosomal membranes in N-ethylmaleimide (NEM) in four groups. These groupswere incubated for 30 min at 2 °C with cocaine100 umol litre"1, isoflurane 8 mmol litre"1, halothane5 mmol litre"1 or no addition (NEM control) andthen an additional 30 min after adding sufficientNEM to achieve final concentrations of 0.1, 1.0 and10.0 mmol litre"1. Control samples were processedidentically except that nothing was added duringeach incubation period. After the second incubation,all groups were diluted with Tris 5 mmol litre"1-NaCl buffer 100 mmol litre"1, pH = 8, and centri-fuged at 30000# for 30 min at 4 °C. Pellets wereresuspended and washed by centrifugation twice.The final pellets were resuspended in 5.0 ml ofbuffer and used in binding assays immediately, asdescribed above. Pilot studies showed completeremoval of the anaesthetics from the membranes bythis washing procedure, as indicated by recovery ofcontrol levels of CFT binding.

PHOTOAFFINITY COMPETITION EXPERIMENTS

We have shown previously that halothane may beused as a photoaffinity ligand [12, 13]. With exposureto UV light, debromination of halothane yields achlorotrifluoroethyl (CFTE) radical [14] whichbinds covalently near to its site of generation [15].Because we were concerned that the binding kineticsof these small molecules to protein sites may be toofast to allow detection with the above NEMprotection experiments (see Discussion), we per-formed photoaffinity competition experiments inwhich CFT binding was evaluated after covalentattachment of halothane to synaptosomes with UVlight. Synaptosomes were prepared as above andseparated into three fractions. Two fractions wereexposed for 50 s to 254-nm light (Oreil low pressureHg calibration lamp, distance 6 mm), in 5-mm path-length quartz cuvettes with continuous stirring; oneof these fractions contained halothane 1 mmol litre"1

(final concentration). The last fraction was a non-exposed control. The fractions were diluted andwashed three times as in the NEM experiments, andused immediately in 3H-CFT binding assays, asdescribed above.

Results

The cocaine analogue CFT bound specifically andwith high affinity to synaptosomal membranesprepared from rat brain. Preliminary experiments(data not shown) demonstrated the well establishedsodium dependence of CFT binding [6]. Because wealso found that 3H-CFT binding was inhibited byTris concentrations greater than 50 mmol litre"1, weused routinely Tris 10 mmol litre"1 in these exper-iments. The binding isotherms in the absence ofanaesthetics demonstrated saturation with an ap-parent mean Kd of 8.0 (SEM 0.8) nmol litre"1 and aBmax of 229 (50) fmol/mg protein, in agreement

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Table 1 Binding variables for 3H-CFT to synaptosomes (mt(SEM) of three experiments, each in triplicate). *P < 0.05,**P < 0.001 compared with controls

Group (nmol litre"1)Bmax Hill(fmol/mg protein) coeffici

Control 8.0 (0.8) 229 (50)Halothane 13.8(1.0)* 219(61)Isoflurane 29.8(12.8)** 387(45)*

0.97 (0.0.96 (0.0.98 (0.

0.020 1

0.015-

0.010-

0.005 -

50 100 150 200

Bound 3H-CFT (pmol)

250

Figure 1 Representative Scatchard plots for binding isothermexperiments: control ( • ) , halothane 1 mmol litre"1 (# ) andisoflurane 1.7 mmol litre"1 (O)- The lines represent leastsquares linear regression of the data.

100 -

c

C

'.a

coo

75 -

50-

25 -

- 2 - 1 0 1

Anaesthetic concn (log mmol litre"1)

Figure 2 Competition experiments for halothane (# ) andisoflurane (O)> expressed as percent of control 3H-CFTbinding (0 anaesthetic) (mean (SEM)). Data were fitted by non-linear least squares to sigmoid plots of approximately — 1 Hillcoefficient and fixed upper limit (100%). K, values are given inthe text.

with published data for this cocaine analogue insynaptosomal preparations [7]. The results weredifferent for the two anaesthetics studied. Halothanesignificantly decreased only the apparent Kd, whileisoflurane altered both Kd and Smax (table 1, fig. 1).Competition experiments with the anaestheticsdemonstrated near complete inhibition of CFT (~1 nmol litre1) binding and an apparent K, of 0.61(0.12) mmol litre"1 for halothane and 0.75(0.12)

- 100-

"ocoo"o

Hal.

Figure 3 Alkylation experiments with N-ethylmaleimide(NEM) 1.0 mmol litre"1. 3H-CFT Binding after treatment ofsamples without NEM (control (c), • ) compared with bindingafter treatment with NEM in the presence of no addition (NA,0) , cocaine 100 umol litre"1 (Coc, • ) , isoflurane 8 mmol litre"1

(Iso., g ) or halothane 5 mmol litre"1 (Hal., 11). Data for NEM0.1 and 10.0 mmol litre"' were similar.

mmol litre"1 for isoflurane (fig. 2). Competitionexperiments with higher concentrations of CFT(6 nmol litre"1) showed essentially an identical K{value for halothane (0.64 (0.10) mmol litre"1),whereas that for isoflurane was slightly higher (1.03(0.66) mmol litre"1) (ns). The larger variation withisoflurane was caused by its consistent ability toincrease 3H-CFT binding at low isoflurane concen-trations and the difficulty of fitting sigmoid curves tothis pattern.

The NEM protection assays demonstrated thatalthough cocaine 100 umol litre"1 during NEM ex-posure (0.1, 1.0 or 10.0 mmol litre"1) significantlyprotected CFT binding sites (fig. 3) from alkylation,neither halothane 5 mmol litre"1 nor isoflurane8 mmol litre"1 offered significant protection com-pared with no addition.

The photoaffinity competition experimentsdemonstrated that although UV exposure alonereduced specific CFT binding to 55.2(3.2)% ofcontrol (non-UV exposed) values, there was asignificant further reduction to 29.8(1.8)% whenthe UV exposure was conducted in the presence ofhalothane 1 mmol litre"1 (P < 0.01 compared withUV-only values) (fig. 4). Because this furtherreduction in CFT binding by halothane and UVlight could also be caused by CTFE free radical-induced damage, independently of covalent occu-pation of the CFT site, we compared the influence ofgeneration of free radicals by UV photolysis of otherphotolabile molecules. Benzoquinone (BQ) and 5-diazouracil (5-DU) form free radicals on exposure toUV light. UV absorbance at 254 nm was used tomatch the concentration of these compounds tohalothane under the assumption that concentrationsthat produce similar absorption at this wavelengthproduce a similar free radical flux. These con-centrations (BQ 5 umol litre"1 and 5-DU10 nmol litre"1) were then tested for inhibition of 3H-

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Cocaine binding and inhalation anaesthetics 823

100-c

T3c

coo

a? ou

Figureof cont hal.not exposed to UV light ( • ) were processed in parallel withthe two other UV-exposed samples. The samples exposed toboth halothane and UV ( • ) demonstrated a further significantloss in 3H-CFT binding compared with those exposed only toUV (S).

CFT binding without photolysis, to assure lack ofcompetition with CFT. No significant inhibition ofCFT binding occurred at these concentrations. Afterphotolysis in the presence of these compounds, nofurther reduction in 3H-CFT binding occurredcompared with UV-exposed controls. In fact, a non-significant trend towards protection from UV-induced damage was observed (110 (7.0)% and 104(5)% for 5-DU and BQ, respectively, comparedwith UV only).

Discussion

The precise mechanism by which cocaine and itsanalogues cause inhibition of the dopamine trans-porter is not known. Although some kinetic studiesdemonstrated competitive inhibition [16, 17], otherssuggested non-competitive kinetics [18]. Also, sitealkylation by NEM [11] and site-directedmutagenesis [19] studies provide more direct evi-dence that the cocaine binding site is allosteric withrespect to the substrate (dopamine) site. Recentcloning and expression of both rat [20] and human[21] dopamine transporters should help to resolvethis issue. It is notable that 92% homology existsbetween dopamine transporters from the two species,permitting reasonable extrapolation of these resultsto the human protein.

The data presented here demonstrated that theinhalation anaesthetic halothane inhibited 3H-CFTbinding with an apparent K, very similar to the IC50for inhibition of dopamine [1] and serotonin trans-port [22]. The inhibition of 3H-CFT binding byhalothane appeared to be competitive by Scatchardanalysis of binding isotherms and the results of thephotoaffinity competition experiments were con-sistent, suggesting overlapping sites for halothaneand CFT. The inhibition of 3H-CFT binding byisoflurane is more complex, having mixed kinetic

characteristics. In addition, the K{ for isofluraneinhibition of dopamine and serotonin transport,about 2.3 mmol litre"1 [1,23] was substantiallyhigher than that for inhibition of 3H-CFT binding,suggesting that the two may not be related, at least inthe case of isoflurane. Unfortunately, the site ofisoflurane binding could not be evaluated withphotoaffinity competition methods because of itsphotostable structure. Similarly, the ability of co-caine to protect the site from halothane photo-labelling would be difficult to interpret because ofthe photolabile nature of cocaine. The complexinhibition of CFT binding by isoflurane may berelated to the biphasic nature of inhibition ofdopamine transport noted previously [1]. An in-crease in CFT binding at low isoflurane concen-trations (ns) was suggested in these studies and isconsistent with the increase in Bmax noted in thepresence of this anaesthetic. A biphasic bindinginfluence of isoflurane has also been observed inother proteins, such as the GABAA complex [24].The mechanism or implication of this complex,presumably allosteric influence of isoflurane, is notyet clear, but it emphasizes that substantialdifferences can exist between the effects of thesesmall molecules and argues against the notion of aunitary mechanism of action of inhalationanaesthetics.

It should be noted that the functional dopamineuptake experiments were performed at highertemperatures (22-37 °C) than our binding studies(2 °C). Although it is well known that gas-buffer andgas-membrane partition coefficients (binding)change dramatically with temperature [25], ourmethod of direct injection and dilution of aqueousanaesthetic stock solutions, combined with evidencesuggesting that buffer-membrane partitioning isstable over a wide temperature range (4-40 °C) [25],implies that binding variables are temperatureindependent. We evaluated this in a small series ofexperiments with two concentrations of each an-aesthetic (halothane 0.11 and 0.83 mmol litre"1;isoflurane 0.66 and 2.0 mmol litre"1) in bindingexperiments at 37 °C and found the percent in-hibition of CFT binding to be well within thestandard error limits (slightly to the left of the mean)of the 2 °C curve. Therefore, the apparent K{ doesnot appear to have a major temperature dependence,as expected.

Previous studies using NEM alkylation suggestedthat certain sulphydryl groups are essential forcocaine binding [11]. Importantly, dopamine wasunable to protect the cocaine site, demonstrating itsallosteric nature. Our experiments demonstratedthat the CFT site was not protected by eitherhalothane or isoflurane, implying that the anaestheticand CFT sites do not overlap, in contrast with thephotoaffinity competition experiments and theScatchard analysis (for halothane). It is not clear,however, if ligands with substantially faster dis-sociation rates than the high-affinity "natural"ligand (e.g. cocaine) would be able to afford siteprotection in this non-equilibrium alkylation assay.The dissociation rate for cocaine is six or sevenorders of magnitude slower than that of halothane, as

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estimated by halothane dissociation rates from serumalbumin by NMR studies [26], and publisheddissociation times for cocaine [6]. This was therationale for conducting the photoafflnity compe-tition experiments, as the creation of a covalent bondprevents dissociation of halothane from its bindingsite.

Results of the photoaffinity competition exper-iments suggested that halothane and CFT sitesoverlap because covalent linkage of the halothanephotolysis product near the equilibrium binding sitefor halothane significantly reduced CFT binding.Quantitation is difficult, however, because photolysisof halothane is inefficient and is not complete in only50 s of UV exposure. Further, because UV lightcauses site destruction independently, longer UVexposures would be difficult to interpret.

Alternative interpretations of the photoaffinitycompetition results are possible. The data could beconsistent with non-overlapping sites. Thedopamine transporter conformational state whichbinds CFT at lower affinity, possibly because ofallosteric binding by the anaesthetic, may nowbecome a "fixed" conformational state because ofcovalent occupancy of that site. However, it seemsunlikely that the covalently bound CTFE group, themost likely adduct formed by photolysis of halothane[14], which lacks the important and bulky bromineatom, would induce the same conformational changeas halothane itself.

The in vivo relevance of anaesthetic competition atthe cocaine binding site is not clear. The high-affinity cocaine (CFT) binding sites investigated inthis study are thought to be located on catecholaminetransporters; transmembrane proteins which areresponsible for termination of catecholamine trans-mission by reuptake into presynaptic terminals.Inhibition of such transporters by cocaine and itsanalogues increases the synaptic concentration ofcatecholamines and presumably enhances suchtransmission. This is thought to be the mechanism ofcocaine-induced euphoria [6], especially as it relatesto dopaminergic transmission. It is difficult to knowif anaesthetics cause euphoria, as at these con-centrations (2-3 times MAC) they also produceunconsciousness, presumably because of actions atother neuronal targets. However, it is attractive tospeculate that an important side effect of halothane,its cardiac arrhythmogenic potential [27], is causedby this cocaine-like influence on catecholaminetransport in the heart, especially as the K, for CFTbinding is similar to the ED50 for halothane (formovement), approximately 0.3 mmol litre"1. Also, itis well known that isoflurane has a significantly lowerpotential to produce arrhythmia and it is a lesspotent inhibitor of dopamine [1] or serotonin [23]transport and CFT binding (this study). Furtherstudies of noradrenaline transport are required toexamine this question. Finally, the suggestion ofoverlapping sites for halothane and CFT, and thedifferent effects of the two agents, support the notionthat at least some effects of inhalation anaestheticsare mediated by direct interaction with protein andthat this influence is different for different inhalationanaesthetics.

AcknowledgementsWe thank Dr M. E. Eldefrawi, Dr E. A. El-Maghrabi for tech-nical assistance and Drs J. M. Gonzales and M. E. Eckenhoff forreview of this manuscript. Supported by GM-46724 and aFoundation for Anaesthesia Education and Research YoungInvestigator Award.

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