Intravenously administered anesthetics directly inhibit airway smooth muscle contraction. Because many anesthetic agents affect membrane ion channel function and sustained contraction of airway smooth muscle requires the influx of Calcium2+ through voltage-dependent Calcium2+ channels, it was hypothesized that intravenous anesthetics inhibit airway smooth muscle voltage-dependent Calcium2+ channels.
Methods: Porcine tracheal smooth muscle cells were enzymatically dispersed and studied using whole-cell, patch-clamp techniques. The cells were exposed to thiopental (10 sup -7 - 3 x 10 sup -4 M), ketamine (10 sup -6 - 10 sup -3 M), or propofol (10 sup -7 - 3 x 10 sup -4 M) while recording macroscopic voltage-activated Calcium2+ currents (ICa).
Results: Each intravenous anesthetic tested significantly inhibited ICain a dose-dependent manner with 3 x 10 sup -4 M thiopental, 10 sup -3 M ketamine, and 3 x 10 sup -4 M propofol each causing approximately 50% depression of peak ICa, but with no apparent shift in the voltage dependence of induced ICa. After pretreatment with the Calcium2+ channel agonist Bay K 8644, thiopental, but not ketamine or propofol, shifted the maximum ICato more positive potentials. All three anesthetics promoted the inactivated state of the channel at more negative potentials, but propofol was less effective than thiopental or ketamine in this regard.
Conclusions: Three intravenous anesthetics evaluated in this study decreased the ICaof porcine tracheal smooth muscle cells but with subtle electrophysiologic differences. Hence, thiopental, ketamine, and propofol each inhibit L-type voltage-dependent Calcium2+ channels of porcine tracheal smooth muscle cells but the molecular mechanisms involved may be agent specific. This inhibition may contribute to the airway smooth muscle relaxant effects of these agents observed in vitro at concentrations greater than those encountered clinically. (Key words: Anesthetics, intravenous: ketamine; propofol; thiopental. Channels, ions; calcium. Muscle, smooth: trachea.)
INTRAVENOUS anesthetics, especially ketamine, are known to cause bronchodilatation and to inhibit bronchoconstriction. This effect may be achieved directly by relaxing smooth muscle cells of the airway and/or indirectly by blocking airway reflexes. For example, ketamine has been shown both to inhibit the excitability of the vagus nerve and to relax airway smooth muscle preparations. The effects of thiobarbiturates are more complex. Thiobarbiturates inhibit vagus nerve reflexes but may either contract or relax airway smooth muscle, depending on the dose, on the condition of preconstriction, and on the species studied. Although it has been shown that the contractile effect of thiopental on airway smooth muscle in vitro was mediated by constrictor prostaglandins, the inhibitory mechanism of this anesthetic is still unknown. Recently, propofol (2,6-diisopropylphenol), a new short-acting intravenous anesthetic, was reported to antagonize fentanyl-induced bronchoconstriction during surgery, to inhibit postoperative bronchospasm in patients with hyperreactive airway disease, and to decrease the incidence of wheezing in patients after induction of general anesthesia and tracheal intubation. The mechanism of the direct inhibitory effect of this anesthetic on airway smooth muscle has not yet been determined. .
Because the concentration of intracellular free Calcium2+([Ca2+]i) plays a central role in the regulation of airway smooth muscle tone, a possible mechanism for relaxation by intravenous anesthetics is a decrease in [Ca2+]i. Furthermore, sustained contraction of airway smooth muscle requires the continued entry of extracellular Calcium2+and blockade of voltage-dependent Calcium2+ channels (VDCs) suppresses the sustained increase in [Ca sup 2+]iin agonist-stimulated tracheal smooth muscle. Hence, we hypothesized that intravenous anesthetics attenuate airway smooth muscle contraction by inhibition of VDCs. To test this hypothesis, we used patch-clamp techniques to directly measure the effects of the intravenous anesthetics thiopental, ketamine, and propofol on inward Calcium2+ currents through VDCs (ICa) in porcine tracheal smooth muscle cells.
Methods and Materials
Preparation of Dispersed Porcine Tracheal Smooth Muscle Cells
This study was approved by the Johns Hopkins Medical Institutions Animal Care and Use Committee. Pigs (Sus scrofa, weighing 30–45 kg) were sedated with 25 mg/kg intramuscular ketamine, anesthetized with 7–8 mg/kg intravenous pentobarbital, and exsanguinated. Tracheas were excised quickly and placed in modified Krebs solution equilibrated with 95% O2-5% CO2at 4 degrees Celsius (composition in mM: NaCl 118, KCl 4.7, NaHCO321, MgSO41.2, KH2PO41.2, glucose 10, and CaCl22.5; pH approximately 7.4). Cell dispersion was performed according to previously described methods. Briefly, tracheal smooth muscle was minced and incubated for 10 min in Calcium2+-free modified Tyrode's solution at room temperature (22–24 degrees Celsius). The modified Tyrode's solution contained (in mM): NaCl 135, KCl 5.4, MgCl sub 2 1.0, glucose 5.0, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] 5.0, and 0.1%(w/v) bovine serum albumin; pH adjusted to 7.4 with 0.5 M tris-[hydroxymethyl]aminomethane. The tissue was then digested for 25 min at 37 degrees Celsius in Calcium2+-free modified Tyrode's solution with added 0.08%(w/v) collagenase, 0.05% trypsin inhibitor, and 0.03% protease. Cells were dispersed by trituration, filtered through nylon mesh, and centrifuged. The pellet was resuspended in a modified Kraftbruhe solution and stored at 4 degrees Celsius for up to 5 h before use. The modified Kraftbruhe solution contained (in mM): KCl 85, K sub 2 HPO430, MgSO45.0, Na2ATP 5.0, pyruvic acid 5.0, creatine 5.0, taurine 20, beta-hydroxybutyrate 5.0, and 0.1%(w/v) fatty acid-free bovine serum albumin; pH adjusted to 7.25 with tris-[hydroxymethyl]aminomethane.
Whole-cell Patch Clamp Recording
All experiments were performed at room temperature (22–24 degrees Celsius). Micropipettes were pulled from soda-lime hematocrit tubing (Fisher Scientific, Pittsburgh, PA) and had resistances of 4–6 M Omega when filled with solution. The pipette solution contained (in mM): CsCl 130, MgCl24.0, EGTA 10, Na2ATP 5.0, and N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] 10; pH adjusted to 7.2 with tris-[hydroxymethyl]aminomethane. The bath solution contained (in mM): tetraethylammonium chloride 130, MgCl21.0, CaCl210, glucose 10, and N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] 10; pH adjusted to 7.4 with tris-[hydroxymethyl]aminomethane.
An aliquot (approximately 0.5 ml) of the cell suspension was placed in a perfusion chamber on the stage of an inverted microscope (Olympus CK2, Leeds Instruments, Minneapolis, MN). A patch pipette was placed against the membrane of a tracheal smooth muscle cell using a three-dimensional oil-driven micromanipulator (MO-102, Narishige, Tokyo, Japan). After obtaining a high-resistance seal (3–20 G omega) with slight suction, the patch membrane was disrupted by strong negative pressure. Membrane currents were monitored using a List EPC-7 patch clamp amplifier (Medical Systems, Greenvale, NY) and the amplifier output was low-pass filtered at 900 Hz. All data were digitized (2,000 samples/s), stored on a hard disk, and analyzed later.
Whole-cell inward Calcium2+ currents (ICa) were elicited at 5-s intervals by 50 ms depolarizing pulses (-50 to +40 mV) from a holding potential of -80 mV. Leak and capacitative currents, estimated by appropriate scaling of currents during 20-mV hyperpolarizing pulses, were subtracted from each of these records. Series resistance was not compensated. Inactivation curves were determined using a double-pulse protocol that consisted of a 1-s duration prepulse to a potential in the range -80 to +20 mV, followed by a 50-ms depolarization to +20 mV. The peak change in current during the test pulse was expressed as a fraction of that obtained with the -80 mV prepulse and this quantity was least-squares fitted to a Boltzmann expression to estimate the potential of half-maximal inactivation (V1/2) and the slope factor (kappa).
Voltage-pulse protocols were performed in control solutions for > 5 min to obtain a stable baseline. Data from cells that showed unstable ICaamplitudes, < 100 pA of peak ICa, or a > 10% reduction in amplitude during the control recording period were discarded. In some experiments, the Calcium2+ channel agonist Bay K 8644 was added 5 min before exposure to anesthetic. Cells were then exposed to a single concentration of one of three intravenous anesthetics (thiopental, 10 sup -7 - 3 x 10 sup -4 M;, ketamine, 10 sup -6 - 10 sup -3 M; or propofol, 10 sup -7 - 3 x 10 sup -4 M) by changing the inflow perfusate of the chamber to one of similar composition but with the anesthetic. Replacement of the chamber solution (approximately 3 ml/min) required approximately 1 min. After a 5-min exposure, the perfusate was reswitched to the control solution. The G omega-seal was maintained for a period sufficient to evaluate the reversibility of anesthetic effects in 61 of 75 experiments (81%).
The following drugs and chemicals were used: type I-S trypsin inhibitor (from soybean), bovine serum albumin, Na2ATP, pyruvic acid, creatine, taurine, beta-hydroxybutyrate, ketamine hydrochloride (Sigma Chemical, St. Louis, MO), type-I collagenase (Gibco Laboratories, Grand Island, NY), protease, Bay K 8644 (Calbiochem, La Jolla, CA), sodium thiopental (Pentothal; Abbott Laboratories, North Chicago, IL), propofol (Diprivan; Stuart Pharmaceuticals, Wilmington, DE), and Intralipid 10%(Kabi Vitrum, Alameda, CA). Bay K 8644 was dissolved in ethanol (0.01% final concentration). Sodium thiopental (from 9.5 x 10 sup -2 M solution in 15 mM Na2CO3) had no effect on pH of the bath solution within the range 10 sup -7 to 3 x 10 sup -4 M. Propofol was diluted from an aqueous emulsion (5.6 X 10 sup -2 M) in 10%(v/v) soybean oil, 2.25% glycerol, and 1.2% purified egg lecithin.
Data are expressed as mean plus/minus SEM. Changes in peak ICaor in the inactivation parameters V1/2 and kappa with exposure to each drug were compared at each applied potential by the paired, two-tailed t test. The percent of control peak ICaand the values of V1/2 and kappa after treatment were compared between anesthetics at equi-effective concentrations using one-factor analysis of variance and Fisher's a posteriori test. In all comparisons, P < 0.05 was considered significant.
Characteristics of Calcium Currents
As previously reported, ICaseen in enzymatically dispersed porcine tracheal smooth muscle cells during step depolarizations from -80 mV peaked at approximately 10 ms and slowly inactivated (Figure 1(A)). Under baseline conditions, threshold activation of ICaoccurred at approximately -20 mV and maximum peak current amplitude was obtained at approximately +20 mV. In 45 cells, the maximum peak ICawas -301 plus/minus 3 pA (range -178 to -509 pA). As shown in a representative trace for depolarization from -80 to +10 mV (F1-18(A)), Bay K 8644 (10 sup -6 M) enhanced the magnitude of ICabut did not appear to alter the time course of the currents. Bay K 8644 significantly enhanced ICaat step potentials in the range -30 to +40 mV and increased the maximum peak ICafrom -319 plus/minus 18 pA at +20 mV to -656 plus/minus 60 pA at +10 mV (approximately 2.1 fold, n = 5). There was a approximately 10 mV shift of the peak ICaversus applied potential curve toward more negative potentials (F1-18(B)). Inward currents with a similar time course were observed in the inactivation experiments. The inactivation parameters obtained in 15 cells under control conditions were V1/2 =-19.9 plus/minus 0.3 mV and kappa = 7.2 plus/minus 0.2 mV.
Effects of Intravenous Anesthetics on the Activation of Macroscopic Voltage-activated Calcium sup 2+ Currents
As shown in a representative trace for depolarization from -80 to +20 mV (Figure 2(A)), thiopental (3 x 10 sup -4 M) inhibited the magnitude of ICabut did not obviously alter the time course of the currents. Peak ICaobtained with repeated steps to +20 mV increased over a few minutes after obtaining the whole cell configuration at time 0 to a stable plateau, decreased rapidly approximately 50% during exposure to 3 x 10 sup -4 M thiopental, and recovered completely with washout (F2-18(B)). Similar results were obtained with ketamine and propofol. Figure 3shows the relationship between peak ICaversus applied potential before and after exposure to 3 x 10 sup -4 M thiopental, 10 sup -3 M ketamine, or 3 x 10 sup -4 M propofol. Each of these intravenous anesthetics significantly inhibited ICaat step potentials in the range -20 or -10 to +40 mV and decreased the peak ICaat +20 mV by approximately 50%(n = 5). The actual percent inhibitions of peak ICaachieved by these agents at these concentrations (51.5 plus/minus 6.9, 49.2 plus/minus 6.1, and 47.9 plus/minus 3.9, respectively) were not significantly different. There was no apparent shift in the voltage-dependence of induced ICawith any anesthetic.
Dose-dependence of the Inhibition of Macroscopic Voltage-activated Calcium sup 2+ Currents by the Intravenous Anesthetics
We determined the dose-dependence of the inhibition of peak ICaby each of these intravenous anesthetics. Figure 4shows the relationship between the percent of control peak ICaat +20 mV and the concentration of the anesthetic in the bath solution (M). Each of the three intravenous anesthetics significantly inhibited peak ICain a dose-dependent manner. Based on total concentration in the solution, thiopental and propofol had similar potency whereas ketamine required somewhat greater concentrations to achieve the same inhibitory effect.
Effects of Intravenous Anesthetics on Macroscopic Voltage-activated Calcium sup 2+ Currents after Activation with Bay K 8644
Pretreatment with the Calcium2+ channel agonist Bay K 8644 did not prevent the anesthetic-induced inhibition of ICa. Figure 5shows the time course of the peak ICaobtained in a representative cell with repeated steps to +10 mV during exposure to 10 sup -6 M Bay K 8644 and 10 sup -3 M ketamine. Despite a substantial enhancement of the magnitude of peak ICaby Bay K 8644, 10 sup -3 M ketamine still induced a approximately 50% inhibition. Similar results were obtained with 3 x 10 sup -4 M thiopental and 3 x 10 sup -4 M propofol in the presence of 10 sup -6 M Bay K 8644. However, thiopental caused a shift in the relationship between peak ICaand applied voltage to more positive potentials. The maximum value occurred at +10 mV in the presence of Bay K 8644 and +30 mV in the presence of both Bay K 8644 and thiopental. This change in voltage dependence was not seen in the absence of Bay K 8644 (F3-18) or with either of the other two anesthetics tested.
Effects of Intravenous Anesthetics on Steady-state Inactivation of Macroscopic Voltage-activated Calcium sup 2+ Currents
The effects of the intravenous anesthetics thiopental, ketamine, and propofol at equi-effective inhibitory concentrations (3 x 10 sup -4 M, 10 sup -3 M, and 3 x 10 sup -4 M, respectively) on the inactivation curves of ICaare summarized in Figure 6and Table 1. Each of these anesthetics shifted the inactivation curve to more negative potentials (F6-18). The induced changes in V1/2 were statistically significant in each case (T1-18). However, the value of V1/2 in the presence of propofol remained significantly more positive than that measured in the presence of either other agent (P < 0.05). The slope factor k was not changed by exposure to any anesthetic. Exposure of five additional cells to the propofol vehicle (0.05% soybean oil, 0.01% glycerol, and 0.006% egg lecithin; from Intralipid 10%) caused no change in the magnitude or voltage dependence of ICaor in the inactivation curve parameters of V1/2 or k (data not shown).
Characteristics of Inward Currents
Using whole-cell, patch-clamp techniques we measured depolarization-induced inward currents in porcine tracheal smooth muscle cells under ionic conditions designed to inhibit Potassium sup + and Sodium sup + currents and to enhance Calcium2+ currents. Based on their time and voltage dependences, their sensitivity to blockade by nifedipine (reported previously ), and their enhancement by the Calcium2+ channel agonist Bay K 8644 (F1-18), these currents are presumed to reflect the activity of L-type VDCs. .
Effects of Intravenous Anesthetics on the Activation of Macroscopic Voltage-activated Calcium sup 2+ Currents
Each of the intravenous anesthetics tested inhibited ICathrough VDCs of porcine tracheal smooth muscle cells without an apparent change in the time course of the currents (F2-18(A)). The onset of inhibition was rapid and the effect was reversible (F2-18(B)) and dose-related (F4-18). None of the intravenous anesthetics altered the voltage dependence of ICa(F3-18). These data demonstrate a cellular effect of intravenous anesthetics that can account for the airway smooth muscle relaxant effects of these agents. The relatively high concentrations of thiopental and ketamine required to inhibit VDCs are similar to those required to directly relax preconstricted airway smooth muscle preparations. Our results are also consistent with data obtained with vascular smooth muscles in which indirect evidence has suggested inhibition of VDCs by barbiturates and propofol and in which ketamine has been shown to inhibit whole-cell patch clamp ICa. .
Effects of Intravenous Anesthetics on Macroscopic Voltage-activated Calcium sup 2+ Currents in the Presence of Bay K 8644
To investigate the molecular basis of intravenous anesthetic effects on VDCs, we performed additional experiments with the Calcium2+ channel agonist Bay K 8644. Bay K 8644 (10 sup -6 M) enhanced ICaapproximately 2 fold and caused a slight shift of the peak ICaversus applied potential curve to more negative potentials (F1-18). However, Bay K 8644 did not prevent inhibition of ICaby thiopental, ketamine, or propofol. Interestingly, thiopental but not ketamine or propofol caused a substantial shift in the voltage dependence of induced ICato more positive potentials. This distinctive action of thiopental indicates that, in addition to physicochemical or non-specific interactions with VDCs, intravenous anesthetics have chemically specific effects on VDC properties. One possible explanation for the additional effect of thiopental is that it interacts with the portion of the channel protein that functions as the voltage sensor for activation.
Effects of Intravenous Anesthetics on the Inactivation of Macroscopic Voltage-activated Calcium sup 2+ Currents
To further examine the inhibitory actions of these intravenous anesthetics on VDCs of tracheal smooth muscle cells, we studied the effects of these anesthetics on steady-state, voltage-dependent inactivation of ICa. During prolonged depolarization a fraction of the VDCs enters an unavailable or "inactivated" state. The degree of steady-state inactivation depends on the pre-pulse potential (F6-18). The mean potential of half inactivation (V1/2 =-19.9 mV) and the mean slope factor (k = 7.2 mV) that we obtained with porcine tracheal smooth muscle cells under baseline conditions are each similar to the values previously reported for canine and bovine tracheal smooth muscle cells. Each of the three intravenous anesthetics tested significantly shifted the inactivation curves to more negative potentials without changing the sigmoid shapes of the curve. A qualitatively similar shift induced by nifedipine in canine colonic smooth muscle cells has been interpreted as evidence for drug-induced stabilization of the inactivated state. In our experiments, the change in V1/2 induced by propofol (6 mV) was significantly less than that by the other two anesthetics (11–12 mV;F6-18and T1-18). However, a vehicle control for propofol had no effect on V1/2. These data are consistent with chemically specific differences in the interaction of intravenous anesthetics with the inactivation mechanism of VDCs.
Concentration Dependence and Clinical Relevance
Although contractile agonists can trigger transient elevations of [Ca sup 2+]iand tension of airway smooth muscle via release from intracellular stores, sustained contraction is dependent on the continued entry of extracellular Calcium2+. A major pathway for Calcium sup 2+ influx during cholinergic contractions is via the VDCs, which give rise to the currents measured in this study. Removal of extracellular Calcium2+ and treatment with Calcium2+ channel blockers are each known to relax airway smooth muscle in vitro under a variety of contractile conditions. Hence, the inhibition of ICaand the promotion of the inactivated state by intravenous anesthetics demonstrated in this study are both effects that would be expected to antagonize bronchoconstriction. However, these effects are of clinical interest only if they occur at concentrations that are usually attained during anesthesia.
Thiopental, ketamine, and propofol each showed concentration-dependent inhibition of ICa(F4-18). Based on the total solution concentration, ketamine was less potent than the other agents by a factor of approximately 3. Because propofol was added as an emulsion it is likely that its free concentration was substantially less than the total and that this drug is, in fact, more potent than thiopental for inhibition of ICa. We suggest that the potency order of these agents is propofol > thiopental > ketamine. Extrapolation of our data to the clinical situation must be viewed with caution because of possible species differences, in vivo/in vitro differences, and the fact that our patch clamp experiments were carried out under nonphysiologic conditions of low (ambient) temperature and high (10 mM) extracellular Calcium2+ concentration. Nonetheless, the peak plasma concentration of thiopental during induction of general anesthesia in humans is typically 5 x 1010- to 3 x 10 sup -4 M. Because of its high affinity to plasma proteins (approximately 90% bound) the free plasma concentration of thiopental is less than 5 x 10 sup -5 M. Plasma concentrations for ketamine during surgical anesthesia are 2–5 x 10 sup -6 M with approximately 12% bound to plasma proteins, whereas those for propofol are 2–5 x 10 sup -5 M with 97–99% bound to proteins. Thus the free concentrations of these drugs in solution, which are required to inhibit airway smooth muscle VDCs (F4-18), would appear to be substantially higher than the free concentrations observed clinically in serum.
In summary, thiopental, ketamine, and propofol each decreased ICaof porcine tracheal smooth muscle cells indicating inhibition of VDCs. This response can account for the ability of these agents to relax airway smooth muscle in vitro. Measurements of anesthetic effects on the voltage dependences of depolarization-induced ICaand of inactivation revealed electrophysiologic differences among the agents and suggested that structurally specific interactions contribute to the modulation of VDC function. Hence, a variety of intravenous anesthetics inhibit L-type VDCs of airway smooth muscle cells but the molecular mechanisms involved are probably agent specific. Unlike inhalational anesthetics, the concentrations of intravenous anesthetics required to inhibit VDCs likely exceed those obtained clinically. Thus, other effects of these agents such as inhibition of neural reflexes are more likely to be responsible for their bronchodilatory effects in vivo.
The authors thank Judy Clancy, for technical assistance.