Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro

J. A. Gredell1, P. A. Turnquist2, M. B. MacIver2 and R. A. Pearce1,*

1 Department of Anesthesiology, University of Wisconsin, Madison, WI 53706, USA. 2 Department of Anesthesia, Stanford University, California, USA

* Corresponding author. E-mail: rapearce{at}wisc.edu

Accepted for publication July 22, 2004.


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Propofol (2,6-diisopropylphenol) is a widely used general anaesthetic that modulates {gamma}-aminobutyric acid type A (GABAA) receptors, the major inhibitory neurotransmitter receptor in the brain. Previous studies have found that the concentration of propofol that is required to affect synaptic inhibition in brain slices is much higher than the free concentration that is achieved clinically and that modulates isolated receptors. We tested whether this is accounted for by slow equilibration in brain tissue, and determined the concentration that must be applied to achieve appropriate brain levels.

Methods. Rat brain slices 300-µm thick were placed in a solution of 100 µM propofol in artificial cerebrospinal fluid for times ranging from 7.5 to 480 min. Concentrations in these slices were measured by HPLC to determine diffusion and partition coefficients. Electrophysiological measurements of the rate at which effects of 5 µM propofol developed were compared with the calculated rate of increase in tissue concentration.

Results. The diffusion coefficient was approximately 0.02x10–6 cm2 s–1, and the brain:artificial cerebrospinal fluid partition coefficient was 36. Diffusion times in brain slices agreed well with time course measurements of propofol-induced depression of synaptic responses, which continued to increase over 5 h. This depression was reversed by blocking GABA inhibition with picrotoxin (100 µM).

Conclusions. Propofol does enhance inhibition in brain slices at a concentration of 0.63 µM in the superfusate, which produces brain concentrations corresponding with those achieved in vivo, but equilibration requires several hours. It is likely that slow diffusion to GABA receptors accounts for the high concentrations (>10 µM) that were needed to depress evoked responses in previous investigations.

Keywords: anaesthetics, i.v., propofol ; pharmacokinetics, propofol ; brain, GABA ; brain, hippocampus ; measurement techniques, electrophysiology


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Propofol (2,6-diisopropylphenol) is a highly lipid-soluble general anaesthetic that is commonly used for the induction and maintenance of anaesthesia and for sedation in the intensive care unit. Like many other general anaesthetics, propofol enhances {gamma}-aminobutyric acid type A (GABAA) receptor function.1 2 Depending on the concentration of propofol, several types of effects are observed, including modulation of GABA-evoked responses at low concentrations (~1 µM), direct channel activation at intermediate concentrations (~10 µM), and channel block at high concentrations (~100 µM).3 4 These effects are thought to contribute to sedation, amnesia, unconsciousness, and immobility,57 and possibly to other aspects of the anaesthetic state. However, propofol has also been reported to alter neurotransmitter release via mechanisms independent of GABAA receptors,8 and to suppress the activity of several other types of ion channels and enzymes.915 It remains unclear to what extent the different actions on GABAA receptors and on other targets contribute to its sedative, memory-impairing, or other anaesthetic properties and side-effects.

To clarify the relative roles of different putative drug targets that are identified using biochemical techniques or reduced preparations it is useful to study drug effects in more intact systems. Towards this end, brain slice preparations have been developed for anaesthetic studies.1618 In addition to the mechanical stability and intact circuitry that such preparations afford, they offer a number of other advantages over in vivo electrophysiological techniques. These include the ability to precisely control the ionic milieu and to deliver pharmacological agents at known concentrations. Using brain slice recordings to assess the effects of propofol in the hippocampus, concentrations between 7 and 28 µM were required to enhance GABAA receptor-mediated recurrent inhibition,19 and the apparent EC50 for slowing inhibitory postsynaptic current decay was 11.4 µM.20 In contrast, a widely quoted estimate for the free concentration that produces anaesthesia is only 0.4 µM,21 and effects on GABAA receptors expressed in cultured cells are observed at comparably low concentrations.4 22 23 A possible explanation for this discrepancy, and for the generally high concentrations required in other brain slice studies,24 is that the tissue concentrations that are achieved within the time frame of typical experiments may be lower than equilibrium concentrations because of slow equilibration. For most drugs that are applied via superfusion, steady-state effects are achieved over several minutes. However, for some drugs, particularly highly lipid-soluble and aqueous-insoluble agents, equilibration times can be substantially longer.25 26 Propofol may fit into this latter category. Indeed, it was reported that suppression of stimulated dopamine release in brain slices continued to increase without reaching a plateau over a 2-h period,24 and its effect on recurrent inhibition recovered by only 10% during a 40-min wash period.19

Therefore, to determine the rate at which propofol equilibrates with brain slice tissue in vitro, and the concentration that must be delivered to achieve concentrations that correspond to those produced by in vivo administration, we applied propofol to brain slices for varying durations up to 480 min and measured the net uptake of drug into the tissue by extraction and HPLC analysis. Application of a one-dimensional diffusion model27 allowed us to estimate the diffusion and partition coefficients of the drug and to determine the drug concentrations within the tissue as a function of depth and time. This analysis showed that depending on the tissue depth at which physiological responses are tested it can take several hours to reach equilibrium. Physiological tests of the effect of propofol on evoked population activity were found to be compatible with such slow equilibration, and further suggested that free concentrations below 1 µM do indeed substantially enhance recurrent inhibition in the hippocampal circuit. Thus, our results indicate that when the rate of equilibration of propofol with brain tissue is taken into account, propofol does alter GABAA receptor-mediated inhibition in brain slices at clinically observed concentrations.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Brain slice tissue preparation
Brain slices were prepared using standard techniques, as described previously.17 28 Briefly, mature Sprague–Dawley rats (P28-P40) were decapitated under isoflurane anaesthesia. The brain was removed and immersed in cold (4°C) artificial cerebrospinal fluid (ACSF) of composition (mM): NaCl 127, KH2PO4 1.21, KCl 1.87, NaHCO3 26, CaCl2 2.17, MgSO4 1.44, glucose 10, saturated with ‘carbogen’ gas (95% oxygen/5% carbon dioxide), pH 7.4. A block of tissue containing the hippocampus was fixed to a tissue tray using cyanoacrylate glue. For electrophysiological experiments, tissue slices 400-µm thick were prepared using a vibrating microtome and stored in carbogen-saturated ACSF at room temperature until use. For uptake measurements, preliminary experiments using 400-µm-thick slices yielded increasing tissue concentrations that did not reach a plateau for over 4 h, so 300-µm-thick slices were prepared for these experiments to permit more rapid equilibration.

Propofol uptake experiments
Single 300-µm-thick brain slices were placed in 200-ml flasks containing ACSF plus 100 µM propofol and 1:1000 dimethyl sulfoxide (DMSO) that had been pre-equilibrated with carbogen gas. Flasks were placed in a covered container with a humidified carbogen atmosphere, and the assembly was agitated gently using a clinical rotator (Fisher Scientific, Pittsburgh, PA) to ensure that the slices remained suspended in solution so that drug diffused into both tissue surfaces. Slices remained in solution for time intervals ranging from 7.5 to 480 min, at which time they were removed from solution, blotted to remove excess ACSF, weighed, and transferred to a test tube for extraction and analysis.

Extraction
250 µl of HPLC-grade water and 20 µl of HPLC-grade methanol were added to the brain slice. The sample was vortexed for 30 s, then homogenized for 5 s using a hand-held variable speed homogenizer (Tissue Tearor, Biospec Products, Inc.). 500 µl of HPLC-grade acetonitrile was added, the sample was vortexed for 1 min, then transferred to a 1.5 ml centrifuge tube and centrifuged for 30 min at 4000 g. The supernatant was transferred to a clean 1.5 ml centrifuge tube and stored covered at –20°C overnight before analysis.

Chromatographic instrumentation and sample analysis
Propofol concentrations were determined using an adaptation of the method described by Knibbe and colleagues.29 The HPLC system consisted of a Waters 600 system controller and a Waters 2487 Dual wavelength absorbance detector set at 276 nm (Waters Corporation, Milford, MA). Separation was achieved using a 125x4.0 mm Waters spherisorb column at room temperature, with a mobile phase consisting of acetonitrile, water, and trifluoroacetic acid (60:40:0.1, v/v/v) eluted at 1.5 ml min–1. Samples were thawed at room temperature, and then injected manually with a 50-µl syringe through a Rheodyne 7725i injector port fitted with a 20-µl sample loop. The syringe was cleaned between injections by three rinses with acetone followed by three rinses with the mobile phase. Data were recorded and processed using Waters Millennium32 (version 3.005) software. Each sample was measured in duplicate, and the concentration was taken as the average of the two measurements. The mass of drug in the brain slice was computed from the measured concentration in the injected sample by comparing the propofol peak area from the injected sample to that of a standard curve prepared by adding a known amount of propofol to brain slices of known mass, then subjecting the samples to extraction and analysis. Comparison of propofol concentrations in standard samples vs calibration solutions showed that 61±1.4% of propofol was recovered by the extraction procedure (n=15 samples at three different concentrations).

Data were analysed using MS Excel (Microsoft, Redmond, WA) and Microcal Origin (Microcal Software, Northampton, MA).

Modeling concentration–depth profiles
The concentration–depth profile for diffusion into a brain slice was modelled using a one-dimensional diffusion equation.27 This model was chosen because of the geometry of the brain slices, which were approximately 10x10x0.3 mm thick. The model is given in dimensionless terms by the equation:

(1)
where

(2)

(3)

(4)
D is the diffusivity, x0 is half of the slice thickness (150 µm), x is the depth within the slice, C is the concentration in the slice at time t, and Cf is the equilibrium concentration in the slice.

Electrophysiological experiments
Brain slices were prepared as described above, except that ACSF for incubation and superfusion contained (in mM): NaCl 124, NaH2PO4 1.25, KCl 3.5, NaHCO3 26, CaCl2 2, MgSO4 2, glucose 10, saturated with carbogen gas, pH 7.4. Hemisected brain slices were transferred to a recording chamber and equilibrated for an additional 10 min before electrophysiological recording. ACSF flowed through the recording chamber at a rate of 3.0 ml min–1.

To evoke population responses, Schaffer collateral inputs to hippocampal CA1 pyramidal neurons were activated using bipolar tungsten microelectrodes placed in the stratum radiatum. Glass recording electrodes were used to record evoked field potentials. They were filled with ACSF (2–5 M{Omega}), and placed at a depth of approximately 125–250 µm in the stratum pyramidale to record population spikes, or in the stratum radiatum to record excitatory postsynaptic potentials (EPSPs). Single stimulus pulses (0.01–0.05 ms duration; 10–80 µA at 1.0–5.0 V) were delivered via constant current stimulus isolation units (Grass Instruments, SIU 6D) connected to a Grass S8800 two channel stimulator, at a stimulus rate of 0.05 Hz. Field potential signals were amplified (x1000), filtered (1 Hz to 10 kHz, bandpass), and digitally stored for off-line analysis on a Pentium microcomputer using DataWave (DataWave Technologies, Longmont, CO) or Strathclyde (kindly provided by J. Dempster) electrophysiological analysis software.

Solutions and drugs
Propofol-containing solutions were prepared by diluting a stock solution of 100 mM propofol in DMSO into ACSF to achieve the desired concentration. HPLC calibration solutions were prepared by diluting a solution of 5 mg ml–1 propofol in methanol into mobile phase to yield concentrations of 0.001, 0.01, 0.1, and 0.25 mg ml–1. Glass flasks were used for propofol application to minimize adsorption of propofol during extraction and analysis.3 Salts and reagents were obtained from Sigma/Aldrich, and were of HPLC-grade purity. Propofol was obtained from RBI/Sigma or was a kind gift from Zeneca Pharmaceuticals.

Data analysis
Data were analysed and plotted using Origin (Microcal) and Excel (Microsoft Corporation, Redmond, WA). Fits to mono- or bi-exponential functions were calculated using non-linear least square minimization.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We measured the net rate of propofol uptake into brain tissue by exposing 300-µm-thick brain slices to ACSF containing 100 µM propofol in DMSO (1:1000) for periods ranging from 7.5 to 480 min. The total amount of propofol in each slice was measured by extraction and quantitative HPLC analysis, and expressed as an average tissue concentration by dividing by the mass of the brain slice. Results from a total of 64 such measurements performed on individual brain slices are shown in Figure 1. The data were fit with a monoexponential function, yielding an equilibration time constant of 47.0 min.



View larger version (13K):
[in this window]
[in a new window]
 
Fig 1 Uptake of propofol into brain slices. 300-µm-thick rat brain slices were incubated in ACSF containing 100 µM propofol for durations ranging from 7.5 to 480 min. The concentration in each slice was assessed by extraction and HPLC analysis. Each point represents the average of duplicate measurements from individual slices. The data were fitted by a monoexponential function with a time constant of 47 min.

 
The final brain slice concentration and the rate of uptake were used to determine the brain:ACSF partition coefficient and the diffusion coefficient of propofol in brain tissue. Because the concentration in the brain tissue reached a plateau at 360 min and then declined slightly, perhaps as a result of a change in tissue characteristics during prolonged incubation, the final equilibrium concentration was taken as the average of the values at 360 min. This yielded a final concentration of 618 µg propofol g–1 brain. Taking into account the molecular weight of propofol (178), the density of brain tissue (1.04),30 and the concentration in ACSF (100 µM), this brain concentration corresponds to a brain:ACSF partition coefficient of 36. To estimate the diffusion coefficient, we plotted the uptake data together with the expected average tissue concentrations corresponding to a range of diffusion coefficients, which we calculated using a one-dimensional diffusion model (equation 1). As illustrated in Figure 2, the rate of uptake corresponded to a diffusion coefficient of approximately 0.02x10–6 cm2 s–1.



View larger version (19K):
[in this window]
[in a new window]
 
Fig 2 Determination of the diffusion coefficient for propofol in brain tissue. Average measured tissue concentrations normalized to the concentration at 360 min were plotted together with expected average tissue concentrations corresponding to a range of diffusion coefficients derived from a one-dimensional model of diffusion into both surfaces of a 300-µm-thick tissue slice. Each data point represents mean (SEM) of eight brain slices (from Fig. 1).

 
This diffusion coefficient is quite small, even compared with the diffusion coefficient of other substances that diffuse through tissue slowly, such as the non-immobilizer 1,2-dichlorohexafluorocyclobutane (F6 or 2N, D=0.1x10–6 cm2 s–1).31 For that drug, we showed previously that diffusion is sufficiently slow that at a depth of 125 µm (where extracellular recordings are typically performed), the tissue concentration has reached only 58% of its final equilibrium concentration by the end of a 40-min application.31 This suggests that for similar experiments with propofol it may take several hours to approach an equilibrium concentration, which is much longer than drugs are typically applied to brain slice preparations. To gauge the expected increase in concentration at different depths through a brain slice under typical recording conditions, we used a one-dimensional diffusion model27 to calculate the concentration of propofol as a function of depth and time following a step increase in the surface concentration. The results are shown in Figure 3. Solid lines show calculated concentrations at different depths, and the dashed line shows the average concentration at depths between 25 and 200 µm, which is the location from which electrophysiological recordings are most often obtained. At the end of 300 min, the average concentration has increased to only 63% of the final equilibrium concentration.



View larger version (22K):
[in this window]
[in a new window]
 
Fig 3 Depth–concentration profile of propofol in a 400-µm-thick brain slice. Tissue concentrations at depths from 50 to 400 µm are plotted for a drug with a diffusion coefficient of 0.02x10–6 cm2 s–1 diffusing into one surface of a brain slice (solid lines). Also plotted is the average concentration between depths of 25 and 200 µm (dashed line) and a monoexponential function with a time constant of 145 min and amplitude of 0.75 (dotted line).

 
To test whether physiological responses do indeed develop so slowly, we applied 5 µM propofol to 400-µm-thick brain slices for up to 300 min and recorded extracellular responses to paired-pulse electrical stimulation of the stratum radiatum. Examples of responses under control conditions, after a 300-min application of propofol, and upon addition of 100 µM picrotoxin, are shown in Figure 4A. Under control conditions, electrical stimuli elicited population spikes (downward deflections) superimposed on population EPSPs (upward deflections). Population spikes represent the synchronous firing of pyramidal neurons produced by excitatory synaptic input. The response to the second stimulus is seen to be slightly greater than the first response, a commonly observed pattern that is caused by short-term facilitation of transmitter release from the excitatory nerve terminals. Under the influence of propofol, the response to the first stimulus was depressed by approximately 50%, and the response to the second stimulus was essentially eliminated. These effects were fully reversed by the addition of picrotoxin. The time course over which these effects developed is illustrated in Figure 4B. The amplitude of each response as a fraction of the control response was plotted as a function of time, and the values were fitted by an exponential function. For the first response, the effect of propofol developed with bi-exponential time constants of 19 (45%) and 145 min (55%). The rate of the slow component is compatible with the expected increase in concentration at a depth of 125 µm, which according to other studies is the average depth at which population responses arise in brain slice preparations,31 for a drug with diffusion coefficient of 0.02x10–6 cm2 s–1 if the drug reduces the population spike amplitude by 75% at equilibrium (plotted in Fig. 3, dotted line). The rate of response to the second stimulus developed considerably faster, with a time constant of 14 min.



View larger version (30K):
[in this window]
[in a new window]
 
Fig 4 Slow onset of physiological effects of propofol in the hippocampus. (A) Evoked population spikes in the CA1 stratum pyramidale in response to paired electrical stimulation of stratum radiatum under control conditions, after 5 µM propofol had been applied for 300 min, and following addition of 100 µM picrotoxin. Calibration bar 50 ms, 4 mV. (B) Time course of development of effect of propofol on first (circles) and second (triangles) population spike amplitudes, normalized to the average amplitude during the 20-min control period. Population spike depression was rapidly reversed by the addition of 100 µM picrotoxin.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Because of discrepancies in the literature regarding the relatively higher concentrations of propofol that are required to produce effects in brain slice preparations compared with the concentrations that are effective in reduced preparations,19 20 22 23 together with previous observations indicating that some effects of propofol continue to develop during prolonged application (2 h),24 we examined the rate of equilibration of propofol with brain slices. Our experiments revealed that propofol does diffuse extremely slowly in brain tissue in vitro, its diffusion coefficient being approximately 0.02x10–6 cm2 s–1. For comparison, the diffusion coefficient of halothane is 40-fold greater, approximately 0.8x10–6 cm2 s–1.31 The slow diffusion of propofol means that when propofol is applied to the top surface of a 400-µm-thick brain slice, the average tissue concentration at depths between 25 and 250 µm will have reached only 20% of its final equilibrium concentration at the end of a 30-min application, and only 64% after 300 min (Fig. 3). Our electrophysiological measurements of the effect of propofol were consistent with this derived estimate of equilibration rate. Thus, unlike many or most drugs used in such experiments, one must take into account the time course of equilibration when designing or interpreting experiments with propofol.

Although the diffusion coefficient of propofol in brain tissue is low enough that equilibration is very slow when the drug is applied to the surface of a tissue slice in vitro, when the drug is delivered to the living brain in vivo via intravascular injection then movement into brain tissue can still occur quite rapidly, as evidenced by the rapid onset of action when propofol is used for the induction of anaesthesia. This is due at least in part to the short distances between the microvasculature and tissue sites of action, as the equilibration rate varies with the square of the distance (equation 1). Administration of doses that produce blood levels higher than the EC50 equilibrium concentration may also contribute to a rapid onset of clinical effects.

To estimate the diffusion coefficient of propofol we used 300-µm-thick brain slices and we exposed both surfaces to drug-containing ACSF. These conditions are not typical for physiological experiments, but we did this so that nearly complete equilibration would be achieved before the health of these acutely prepared slices began to deteriorate. Typically, brain slices remain viable for several hours. It would have taken substantially longer than this for complete equilibration had only one surface of a 400-µm-thick slice been exposed. Full equilibration was considered desirable so that we could gauge as accurately as possible the brain:ACSF partition coefficient and the diffusion coefficient. Despite these measures, maximum or near-maximum values were still not reached until drug had been applied for 240 min (Fig. 1). Although we do not have any direct evidence, a probable explanation for the slight reduction between 240 and 480 min is that during this interval the tissue properties changed and tissue integrity was reduced, so that when the brain slice was transferred to the extraction vial for quantitative analysis less that the full amount of tissue (and thus propofol) was included. Indeed, in pilot experiments we found that after 480 min of exposure brain slices began to deteriorate visibly. Therefore, to calculate the partition coefficient we considered the values at 360 min to represent the fully equilibrated condition (Fig. 1), and we used values only up to 360 min to estimate the diffusion coefficient (Fig. 2). If undetected loss of tissue integrity occurred before the 360-min time point, this would have caused us to underestimate the brain:ACSF partition coefficient and overestimate the diffusion coefficient. As we saw no physical evidence of degradation during this time, and in physiological experiments tissue did remain healthy for this duration, we expect any such error to be small.

Drug concentrations that correspond to behavioural effects
Extensive in vivo pharmacokinetic studies have been performed to define the distribution and disposition of propofol in humans and animals. The free concentration that corresponds to the specific end-point of ‘awakening’ in humans, or recovery of the righting reflex in animals, may be estimated from concentrations in blood or plasma together with measurements of partition coefficients and protein binding. The concentration of propofol in whole blood at which rats recover the righting reflex was reported to be 2.832 or 3.5 µg ml–1.33 Utilizing an average ‘awakening’ blood concentration of 3.2 µg ml–1, a whole blood:plasma partition coefficient for rats of 2.3,3436 a fraction of drug bound to plasma proteins of 97.5%,33 and a molecular weight of 178, this corresponds to a free concentration of 0.20 µM. Similar concentrations have been derived from human studies. Spontaneous eye opening occurs at a blood concentration of 2.2 µg ml–1,37 so for a free fraction in human blood of 1.5%,38 this corresponds to a free concentration of 0.19 µM.

How does this estimate of free propofol concentration correspond to brain concentrations that have been measured at different behaviourally or physiologically derived end-points, and to concentrations that we achieved in our experiments?

In sheep, loss of response to a noxious stimulus occurs at brain concentrations of 6–9 µg g–1 brain;39 tail flick latency in rats is greater than 30 s at a concentration of 15.7 µg g–1 brain;36 and in humans, brain concentrations of approximately 17 µg g–1 produce deep anaesthesia and burst suppression.40 Brain concentrations that correspond to awakening or loss of righting reflex have not been measured directly, but may be calculated using previous measurements of blood concentration at awakening and appropriate partition coefficients. Using a value for {lambda}brain:water of 113,30 and a brain density of 1.04, the brain concentration that corresponds to a free concentration of 0.20 µM would be 3.9 µg g–1 brain. This value is reasonable given the concentrations (6–16 µg g–1) that lead to loss of reflex movement. As the partition coefficient that we measured ({lambda}brain:ACSF=36) is only about one-third of the value for {lambda}brain:water estimated by Weaver and colleagues,30 perhaps as a result of a higher solubility of propofol in ACSF plus DMSO than in water, we calculate that superfusion with a solution containing 0.63 µM propofol would lead to an equilibrium brain concentration in vitro of 3.9 µg g–1.

Electrophysiologic effects of propofol in the brain slice
The amplitude of the first evoked population spike was reduced by approximately 50% after propofol had been applied for 300 min (Fig. 4A). At that time, average ‘free concentration’ within the top 250 µm of the slice would have been approximately 3 µM (Fig. 3). The second population spike was essentially eliminated by this concentration of propofol (Fig. 4B). Previous studies have attributed ‘paired-pulse depression’ of the population spike to GABAA receptor-mediated recurrent inhibition.17 41 Thus, the transformation from facilitation to depression may be caused by an enhancement of GABAA receptor function by propofol. Consistent with this hypothesis, subsequent application of picrotoxin caused the response pattern to revert to facilitation (Fig. 4). In addition, the first response returned to its control amplitude, suggesting that its depression by propofol was also mediated by GABAA receptors.

If the inhibitory receptors that underlie propofol effects on the first and second paired responses are present at similar depths within the tissue, a possible explanation for the more rapid effect on the second response is that this effect occurs at a lower concentration than is required for depression of the first response. Examination of Figure 2 reveals that after 10 min of drug application, at which time the first spike was reduced by only 10% and the second spike was reduced by approximately 50%, propofol has reached only 8% of its equilibrium concentration. Thus, it appears that GABAA receptor-mediated recurrent inhibition is substantially enhanced by 0.4 µM propofol (8% of 5 µM). The difference in sensitivity of the first and second responses may reflect differences in the sensitivity of different types of receptors that control the responses, or a difference in the degree of enhancement required to alter the responses. Inhibitory synapses are physiologically heterogeneous in the CA1 region,42 and in addition extrasynaptic receptors that produce a tonic inhibitory current exist.43 These different types of receptors may respond differently to propofol.44 Also, as noted previously, low concentrations of propofol augment responses to GABA but higher concentrations are required to directly activate receptors.3 Thus, it is also possible that the different sensitivity of the two responses depends on the mode of activation of receptors. Further studies to define the molecular receptor-based mechanisms by which propofol alters integrated neuronal responses will be required to address these questions. It will be necessary to take into account the slow equilibration of propofol with brain tissue when designing and interpreting such experiments. These considerations may also apply to other highly lipophilic drugs such as thiopentone and steroid anaesthetics.


    Acknowledgments
 
We thank Dr Onofre DeJesus (Department of Medical Physics, University of Wisconsin) and the UW Department of Chemical Engineering for the use of their HPLC apparatus, and Dr Lisa Birt (Resident in Anesthesiology, University of Wisconsin), and Theresa Foley (undergraduate student, University of Wisconsin Department of Chemical Engineering) for assistance in the development of HPLC protocols. Supported by NIH Grant GM55719 (to R.A.P.), GM54767 (to M.B.M.), and the Departments of Anesthesiology at the University of Wisconsin and Stanford University.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
1 Hales TG, Lambert JJ. The actions of propofol on inhibitory amino acid receptors of bovine adrenomedullary chromaffin cells and rodent central neurones. Br J Pharmacol 1991; 104: 619–28[Abstract]

2 Orser BA, Wang LY, Pennefather PS, MacDonald JF. Propofol modulates activation and desensitization of GABAA receptors in cultured murine hippocampal neurons. J Neurosci 1994; 14: 7747–60[Abstract]

3 Adodra S, Hales TG. Potentiation, activation and blockade of GABAA receptors of clonal murine hypothalamic gt1-7 neurones by propofol. Br J Pharmacol 1995; 115: 953–60[Abstract]

4 Williams DB, Akabas MH. Structural evidence that propofol stabilizes different GABA(A) receptor states at potentiating and activating concentrations. J Neurosci 2002; 22: 7417–24[Abstract/Free Full Text]

5 Nelson LE, Guo TZ, Lu J, Saper CB, Franks NP, Maze M. The sedative component of anesthesia is mediated by GABA(A) receptors in an endogenous sleep pathway. Nature Neurosci 2002; 5: 979–84[CrossRef][ISI][Medline]

6 Krasowski MD, Jenkins A, Flood P, Kung AY, Hopfinger AJ, Harrison NL. General anesthetic potencies of a series of propofol analogs correlate with potency for potentiation of gamma-aminobutyric acid (GABA) current at the GABA(A) receptor but not with lipid solubility. J Pharmacol Exp Ther 2001; 297: 338–51[Abstract/Free Full Text]

7 Jurd R, Arras M, Lambert S, et al. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. FASEB J 2003; 17: 250–2[Free Full Text]

8 Lingamaneni R, Birch ML, Hemmings HC. Widespread inhibition of sodium channel-dependent glutamate release from isolated nerve terminals by isoflurane and propofol. Anesthesiology 2001; 95: 1460–6[ISI][Medline]

9 Orser BA, Bertlik M, Wang LY, MacDonald JF. Inhibition by propofol (2,6 di-isopropylphenol) of the N-methyl-D-aspartate subtype of glutamate receptor in cultured hippocampal neurones. Br J Pharmacol 1995; 116: 1761–8[Abstract]

10 Flood P, Ramirez-Latorre J, Role L. Alpha 4 beta 2 neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but alpha 7-type nicotinic acetylcholine receptors are unaffected. Anesthesiology 1997; 86: 859–65[ISI][Medline]

11 Todorovic SM, Lingle CJ. Pharmacological properties of T-type Ca2+ current in adult rat sensory neurons: effects of anticonvulsant and anesthetic agents. J Neurophysiol 1998; 79: 240–52[Abstract/Free Full Text]

12 Furuya R, Oka K, Watanabe I, Kamiya Y, Itoh H, Andoh T. The effects of ketamine and propofol on neuronal nicotinic acetylcholine receptors and P2x purinoceptors in PC12 cells. Anesth Analg 1999; 88: 174–80[Abstract/Free Full Text]

13 Funahashi M, Higuchi H, Miyawaki T, Shimada M, Matsuo R. Propofol suppresses a hyperpolarization-activated inward current in rat hippocampal CA1 neurons. Neurosci Lett 2001; 311: 177–80[CrossRef][ISI][Medline]

14 Schwieler L, Delbro DS, Engberg G, Erhardt S. The anaesthetic agent propofol interacts with GABA(B)-receptors: an electrophysiological study in rat. Life Sci 2003; 72: 2793–801[CrossRef][ISI][Medline]

15 Haeseler G, Stormer M, Bufler J, et al. Propofol blocks human skeletal muscle sodium channels in a voltage-dependent manner. Anesth Analg 2001; 92: 1192–8[Abstract/Free Full Text]

16 Richards CD, Russell WJ, Smaje JC. The action of ether and methoxyflurane on synaptic transmission in isolated preparations of the mammalian cortex. J Physiol 1975; 248: 121–42[Abstract]

17 Pearce RA. Volatile anesthetic enhancement of paired-pulse depression investigated in the rat hippocampus in vitro. J Physiol (Lond) 1996; 492.3: 823–40

18 MacIver MB, Roth SH. Enflurane-induced burst firing of hippocampal CA 1 neurones. In vitro studies using a brain slice preparation. Br J Anaesth 1987; 59: 369–78[Abstract]

19 Albertson TE, Walby WF, Stark LG, Joy RM. The effect of propofol on ca1 pyramidal cell excitability and GABA(a)-mediated inhibition in the rat hippocampal slice. Life Sci 1996; 58: 2397–407[CrossRef][ISI][Medline]

20 Hollrigel GS, Toth K, Soltesz I. Neuroprotection by propofol in acute mechanical injury: role of GABAergic inhibition. J Neurophysiol 1996; 76: 2412–22[Abstract/Free Full Text]

21 Franks NP, Lieb WR. Molecular and cellular mechanisms of general anaesthesia. Nature 1994; 367: 607–14[CrossRef][ISI][Medline]

22 Jones MV, Harrison NL, Pritchett DB, Hales TG. Modulation of the GABAA receptor by propofol is independent of the gamma subunit. J Pharmacol Exp Ther 1995; 274: 962–8[Abstract]

23 Sanna E, Mascia MP, Klein RL, Whiting PJ, Biggio G, Harris RA. Actions of the general anesthetic propofol on recombinant human GABAA receptors: influence of receptor subunits. J Pharmacol Exp Ther 1995; 274: 353–60[Abstract]

24 Schulte D, Callado LF, Davidson C, et al. Propofol decreases stimulated dopamine release in the rat nucleus accumbens by a mechanism independent of dopamine D2, GABAA and NMDA receptors. Br J Anaesth 2000; 84: 250–3[Abstract/Free Full Text]

25 Dunlap K, Luebke JI, Turner TJ. Identification of calcium channels that control neurosecretion. Science 1994; 266: 828–31[Medline]

26 Thomson AM, Bannister AP, Hughes DI, Pawelzik H. Differential sensitivity to Zolpidem of IPSPs activated by morphologically identified CA1 interneurons in slices of rat hippocampus. Eur J Neurosci 2000; 12: 425–36[CrossRef][ISI][Medline]

27 Bennett CO, Myers JE. Momentum, Heat, and Mass Transfer, 3rd Edn. New York: McGraw-Hill, 1982

28 Lukatch HS, MacIver MB. Voltage-clamp analysis of halothane effects on GABAA,fast and GABAA,slow inhibitory currents. Brain Res 1997; 765: 108–12[CrossRef][ISI][Medline]

29 Knibbe CA, Koster VS, Deneer VH, Stuurman RM, Kuks PF, Lange R. Determination of propofol in low-volume samples by high-performance liquid chromatography with fluorescence detection. J Chromatogr B Biomed Sci Applicat 1998; 706: 305–10[CrossRef][Medline]

30 Weaver BM, Staddon GE, Mapleson WW. Tissue/blood and tissue/water partition coefficients for propofol in sheep. Br J Anaesth 2001; 86: 693–703[Abstract/Free Full Text]

31 Chesney MA, Perouansky M, Pearce RA. Differential uptake of volatile agents into brain tissue in vitro. Measurement and application of a diffusion model to determine concentration profiles in brain slices. Anesthesiology 2003; 99: 122–30[ISI][Medline]

32 Adam HK, Glen JB, Hoyle PA. Pharmacokinetics in laboratory animals of ICI 35 868, a new i.v. anaesthetic agent. Br J Anaesth 1980; 52: 743–6[Abstract]

33 Cockshott ID, Douglas EJ, Plummer GF, Simons PJ. The pharmacokinetics of propofol in laboratory animals. Xenobiotica 1992; 22: 369–75[ISI][Medline]

34 Yeganeh MH, Ramzan I. Determination of propofol in rat whole blood and plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Applicat 1997; 691: 478–82[CrossRef][Medline]

35 Dutta S, Matsumoto Y, Muramatsu A, Matsumoto M, Fukuoka M, Ebling WF. Steady-state propofol brain:plasma and brain:blood partition coefficients and the effect-site equilibration paradox. Br J Anaesth 1998; 81: 422–4[CrossRef][ISI][Medline]

36 Shyr MH, Tsai TH, Tan PP, Chen CF, Chan SH. Concentration and regional distribution of propofol in brain and spinal cord during propofol anesthesia in the rat. Neurosci Lett 1995; 184: 212–5[CrossRef][ISI][Medline]

37 Servin FS, Bougeois B, Gomeni R, Mentre F, Farinotti R, Desmonts JM. Pharmacokinetics of propofol administered by target-controlled infusion to alcoholic patients. Anesthesiology 2003; 99: 576–85[CrossRef][ISI][Medline]

38 Mazoit JX, Samii K. Binding of propofol to blood components: implications for pharmacokinetics and for pharmacodynamics. Br J Clin Pharmacol 1999; 47: 35–42[CrossRef][ISI][Medline]

39 Ludbrook GL, Upton RN, Grant C, Gray EC. Brain and blood concentrations of propofol after rapid intravenous injection in sheep, and their relationships to cerebral effects. Anaesth Intensive Care 1996; 24: 445–52[ISI][Medline]

40 Ludbrook GL, Visco E, Lam AM. Propofol: relation between brain concentrations, electroencephalogram, middle cerebral artery blood flow velocity, and cerebral oxygen extraction during induction of anesthesia. Anesthesiology 2002; 97: 1363–70[ISI][Medline]

41 Rock DM, Taylor CP. Effects of diazepam, pentobarbital, phenytoin and pentylenetetrazol on hippocampal paired-pulse inhibition in vivo. Neurosci Lett 1986; 65: 265–70[CrossRef][ISI][Medline]

42 Pearce RA. Physiological evidence for two distinct GABAA responses in rat hippocampus. Neuron 1993; 10: 189–200[CrossRef][ISI][Medline]

43 Caraiscos VB, Elliott EM, You T, et al. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by {alpha}5 subunit-containing {gamma}-aminobutyric acid type A receptors. Proc Natl Acad Sci USA 2004; 101: 3662–7[Abstract/Free Full Text]

44 Bieda MC, MacIver MB. A major role for tonic GABAA conductances in anesthetic suppression of intrinsic neuronal excitability. J Neurophysiol 2004; 92: 1658–67[Abstract/Free Full Text]