School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia. E-mail: email@example.com
1The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca2+ concentration ([Ca2+]i) and membrane currents were investigated in neonatal rat intracardiac neurons.
2In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca2+]I, which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca2+]i transients was 28 μM, close to the estimated clinical EC50 (clinically relevant (half-maximal) effective concentration) of thiopental.
3In fura-2-loaded neurons, voltage clamped at −60 mV to eliminate any contribution of voltage-gated Ca2+ channels, thiopental (25 μM) simultaneously inhibited nAChR-induced increases in [Ca2+]i and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by ∼ 40% at −120, −80 and −40 mV holding potential, indicating that the inhibition is voltage independent.
4The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC50 were also shown to inhibit nAChR-induced increases in [Ca2+]i by ∼40%.
5Thiopental (25 μM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca2+]i, indicating that inhibition of Ca2+ release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely.
6Depolarization-activated Ca2+ channel currents were unaffected in the presence of thiopental (25 μM), pentobarbital (50 μM) and ketamine (10 μM).
7In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca2+]i transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions.
Intravenous (i.v.) anaesthetics are known to affect cardiac parameters such as heart rate and cardiac output under clinical conditions and in chronically instrumented animals (Blake & Korner, 1981; Inoue & Arndt, 1982; Akine et al., 2001). The underlying mechanisms are complex and could involve anaesthetic effects on the myocardium, central and peripheral neurons. Interestingly, excitatory neurotransmission in sympathetic ganglia is blocked by i.v. anaesthetics and therefore cannot explain the increases in heart rate observed during i.v. anaesthesia (Nicoll, 1978; Mahmoodi et al., 1980). This effect, however, could in part be due to anaesthetic inhibition of parasympathetic neurons that are involved in the regulation of cardiac function (Inoue & Konig, 1988). For example, it has recently been shown that ketamine inhibits nicotinic excitation in cardiac preganglionic parasympathetic neurons of the nucleus ambiguus of the brainstem (Irnaten et al., 2002). Postganglionic intracardiac neurons have also been shown to modulate heart rate in a nicotine-dependent manner, indicating an involvement of nicotinic acetylcholine receptor (nAChR) channels (Bibevski et al., 2000; Ji et al., 2002). These findings strongly suggest an involvement of nAChRs on intracardiac neurons in the modulation of heart rate during anaesthesia, but i.v. anaesthetic effects on nAChR channels in these neurons have not been studied to date.
On a molecular level, clinically relevant concentrations of i.v. anaesthetics have been shown to target glutamate receptor channels and a superfamily of ligand-gated ion channels including nicotinic ACh, GABAA, glycine and 5-HT3 receptor channels (Franks & Lieb, 1994; Krasowski & Harrison, 1999; Yamakura et al., 2001). In particular, barbiturates and ketamine have also been shown to inhibit several subtypes of neuronal nAChR channels (Andoh et al., 1997; Downie et al., 2000; Flood & Krasowski, 2000; Yamakura et al., 2000; Coates & Flood, 2001; Coates et al., 2001; Kamiya et al., 2001), although the α7 homomer appears less sensitive (Tassonyi et al., 2002). Most of these studies, however, have been carried out in expression systems. This is a drawback because nAChR channels expressed in Xenopus oocytes and mammalian cell lines are not necessarily functionally similar to native nAChR channels in ganglionic neurons (Lewis et al., 1997). In particular, the effects of anaesthetics on the electrophysiological properties of ligand-gated ion channels in native neurons can differ from those in expression systems, as demonstrated for GABAA channels in the absence of the (native) channel modulating GABA-receptor-associated protein (Everitt et al., 2004). Therefore, to relate studies of anaesthetic effects on ion channels in expression systems to physiological function, it is important to carry out studies in functionally relevant native cells.
While the α3 and either the β2 or β4 subunits contribute to the composition of nAChRs in all rat intracardiac neurons, α7 subunits are also present in a majority of these neurons (Poth et al., 1997; Cuevas & Berg, 1998), which can form a highly Ca2+-permeable homomeric channel when expressed in Xenopus oocytes (Bertrand et al., 1993; Tassonyi et al., 2002). Electrophysiological recordings have shown that neuronal nAChR channels in rat intracardiac ganglia have a higher Ca2+ permeability than muscle nAChRs found at the neuromuscular junction (Fieber & Adams, 1991; Adams & Nutter, 1992), and it has recently been shown that nAChR activation in these neurons is followed by an increase in cytoplasmic Ca2+ levels concomitant with an influx of Na+ and Ca2+ ions and a transient membrane depolarization (Beker et al., 2003). These findings suggest that nAChR channels are likely to play a significant role in the intracellular Ca2+ homeostasis of intracardiac neurons.
Given that Ca2+ homeostasis is crucial for the regulation of various neuronal functions including membrane excitability, neurotransmitter release and gene transcription (reviewed in Berridge, 1998), it is of interest to determine if anaesthetics alter Ca2+ signalling. In addition to nAChR channels, the activation of voltage-dependent Ca2+ channels, muscarinic ACh receptors (mAChRs) and purinergic (P2) receptors increase [Ca2+]i in rat intracardiac neurons (Liu et al., 2000; Beker et al., 2003). Although voltage-gated Ca2+ channels and purinergic P2X receptor channels mediate Ca2+ entry, these channels appear to be relatively insensitive to i.v. anaesthetics (Franks & Lieb, 1994; Andoh et al., 1997; Hirota et al., 2002). The activation of mAChR and P2Y receptors has been shown to initiate Ca2+ release from inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores (Liu et al., 2000; Beker et al., 2003), whereas ryanodine receptor (RyR) channels have been shown to amplify nAChR-induced Ca2+ transients via Ca2+-induced Ca2+ release (CICR) in these neurons (Beker et al., 2003). However, little is known about the effects of these anaesthetics on intracellular Ca2+ release channels in neurons. In rat papillary muscles, thiopental may inhibit ryanodine-induced Ca2+ release from the sarcoplasmic reticulum (SR) (Komai & Rusy, 1994), whereas in rat smooth aortic muscle, thiopental has been shown to induce Ca2+ release from the SR (Mousa et al., 2000).
The present study investigates the effects of thiopental, pentobarbital and ketamine on parasympathetic neurons of intrinsic cardiac ganglia. Given that modulation of nAChRs in autonomic neurons may contribute to cardiovascular side effects of i.v. anaesthetics, it was of interest to determine if i.v. anaesthetics affect nAChR-activated membrane currents and cytoplasmic Ca2+ levels in rat intracardiac ganglion neurons. A preliminary report of some of these results has been published previously in abstract form (Weber et al., 2002).
Parasympathetic neurons from rat intracardiac ganglia were isolated and placed in tissue culture as described previously (Fieber & Adams, 1991). Briefly, Wistar rats (3–8 days old) were killed by cervical dislocation in accordance with guidelines of Animal Experimentation Ethics Committees of the University of Queensland and the University of Heidelberg. The heart was excised and transferred into dissection solution and atria were removed, placed into dissection solution containing collagenase (0.8 mg ml−1) and incubated for 1 h at 37°C. After the enzymatic treatment, clusters of ganglia were dissected, transferred to a culture media containing sterile culture dish and triturated using a Pasteur pipette with a narrow, fire-polished opening. The isolated neurons were plated on laminin-coated glass cover slips and incubated at 37°C for 24–48 h under a 95% air, 5% CO2 atmosphere. Experimental data presented were obtained from >100 cells dissociated from intracardiac ganglia dissected from 43 neonatal rats.
Absorption spectra measurements
Experiments were performed to determine whether thiopental interferes with the absorption characteristics of the ratiometric Ca2+-sensitive fluorescence indicator, fura-2. Absorption spectra of standard Ca2+-EGTA solutions containing either 0 (control) or 25 μM thiopental, and 0 or 10 μM fura-2 pentapotassium salt were recorded with a UV/vis spectrophotometer (Beckman DU 640, Beckman Instruments Inc., Fullerton, CA, U.S.A.) with a wavelength range from 190 to 1100 nm, a spectral excitation bandwidth of 1.8 nm and a silicon diode for the detection of the transmitted light.
Thiopental (25 μM) slightly increased the absorbance of solutions both in the absence and presence of 10 μM fura-2, indicating the presence of a minor unspecific (not dye-related) thiopental effect. To eliminate the nonspecific effect of thiopental on Ca2+-EGTA solutions in general (in the absence of the dye), the absorbance of the dye-free solutions was subtracted from that of dye-containing solutions. The resulting absorbance curves were virtually identical for solutions in the absence and presence of thiopental, over the complete wavelength spectra of relevance for fura-2 excitation, indicating that thiopental did not interfere with the absorption characteristics of fura-2.
Intracellular [Ca2+] in response to the application of ACh or caffeine was determined in fura-2-loaded rat intracardiac neurons using single-cell ratiometric photometry. Neurons were incubated for 1 h at room temperature in fura-2-loading solution. Subsequently, they were washed in physiological salt solution (PSS) and a recovery period of ∼30 min before experiments was used. Isolated neurons were selected for the experiments to minimize synaptic contacts and the activation of nearby cells. Fura-2-loaded cells were illuminated with light from a 75 W xenon arc lamp, which was split by an optical chopper (OC-4000 Optical Chopper, Photon Technology International (PTI), South Brunswick, NJ, U.S.A.) and passed alternately through 340 and 380 nm band-pass filters. A 510 nm band-pass emission filter was used and a variable aperture set around the cell image. The emitted light was collected by a Hamamatsu R 928 photomultiplier tube, the output of which was digitized using a PTI interface and sampled at 5 Hz using Felix 1.1 software (PTI) run on a 133 MHz computer. Some experiments were carried out on a ratiometric imaging system based on an Olympus IX 70 microscope with UV optics, a Polychrome II monochromator (Till Photonics, Gräfeling, Germany) alternating between 340 and 380 nm illumination, a Hamamatsu C3077 CCD camera with a C2400-80 intensifier head to collect the fluorescence images, a Meteor II frame grabber board (Matrox, Dorval, Quebec, Canada) and Simple PCI 5.0 (Compix Inc., Imaging Systems, Cranberry Township, PA, U.S.A.) software run on a 1600 MHz computer.
Changes in intracellular [Ca2+] (Δ[Ca2+]i) were obtained measuring the ratio of the intensity of the emitted 510 nm fluorescence R(F340/F380) when the cell was illuminated with 340 nm light (F340) to that when illuminated with 380 nm light (F380) and converting this ratio to approximate Ca2+ concentrations using the equation:
where Rmin and Rmax are the F340/F380 ratios of the Ca2+-free and Ca2+-saturated fura-2 sample, respectively, Sf2 is F380 of the Ca2+-free fura-2 sample and Sb2 is F380 of the Ca2+-bound sample. An 11-step in vitro calibration procedure during which [Ca2+] was increased from approximately 0 to saturation of the dye at > mM [Ca2+] was used to determine the numerical values for the constants in this equation using fura-2 pentapotassium salt and standard Ca2+-EGTA solutions (Grynkiewicz et al., 1985). Under the experimental conditions of the PTI set-up, the dissociation constant Kd was determined as 178.9 nM.
A concentration–response curve with thiopental as antagonist for nAChR activation was obtained by measuring the peak increase in [Ca2+]i (Δ[Ca2+]i) at each antagonist concentration and the experimental data points were fitted using the equation:
where Δ[Ca2+]i/Δ[Ca2+]i(max) represents the relative peak increase in Δ[Ca2+]i, [A] is the antagonist concentration, IC50 is the concentration giving half-maximal inhibition and n is the Hill coefficient.
To exclude any thiopental quenching of the ratiometric fura-2 signal, standard calibration procedures were carried out on the ratiometric imaging set-up both in the absence (control: Rmin=0.738±0.003, Rmax=1.411±0.026, Kd=196±7 nM (n=8)) and presence of 25 μM thiopental (Rmin=0.735±0.002, Rmax=1.410±0.017, Kd=190±4 nM (n=8)). None of these values was significantly different from those obtained under the control condition (P<0.3 for all values), indicating that 25 μM thiopental does not interfere with the spectral characteristics of fura-2.
Membrane currents were measured with the whole-cell recording method of the patch-clamp technique, using either the conventional, dialysed (Hamill et al., 1981) or the perforated patch (Horn & Marty, 1988) recording configuration. For perforated patch recordings, amphotericin B-containing solutions were used (Beker et al., 2003). The pipette was first tip-filled with an antibiotic-free solution to prevent disruption of seal formation and then backfilled with the amphotericin B-containing solution. No systematic differences were observed between dialysed and perforated patch recording configurations. Pipettes were pulled from thin-walled borosilicate glass (150TF; Harvard Apparatus Ltd, Edenbridge, Kent, U.K.) using a Sutter Instruments P-87 pipette puller, fire polished and had resistances of ∼2.5 MΩ.
Filled patch pipettes were mounted on a pipette holder connected with the head stage of a patch-clamp amplifier (EPC-7, List-Medical, Darmstadt, Germany or RK300, Bio-Logic, Claix, France). Voltage protocols were applied using Clampex software (Version 8.0, Axon Instruments Inc., Union City, CA, U.S.A.). Calcium channel currents were elicited by step depolarization from −100 mV to −20 mV at 10 s intervals and a −P/4 pulse protocol was employed to subtract leak and transient capacitive currents. Series resistance compensation was routinely used in recording of Ca2+ channel currents and the access resistance was usually <2 MΩ following compensation. Currents were filtered at 5 kHz and sampled at 20 kHz using the Digidata 1200 interface (Axon Instruments Inc.). Agonist-evoked currents were filtered at 200 Hz, digitized at 1 kHz and stored on the hard disc of a Pentium PC for further analysis.
Solutions and drugs
Solutions for absorption measurements and for the calibration procedure of the fura-2 signal were standard Ca2+-EGTA solutions containing 10 mM EGTA, 100 mM KCl, 10 mM K-MOPS and either 10 mM CaCl2 (Ca2+-EGTA solution) or no added Ca2+ (EGTA solution), respectively (Grynkiewicz et al., 1985). The free [Ca2+] of the solution was <0.1 mM in the Ca2+-EGTA solution and approximately zero in the EGTA solution. The dissection solution contained (mM): 140 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 7.7 glucose and 10 histidine (pH adjusted to 7.2 with NaOH). The culture media was high glucose Dulbecco's modified Eagle's media, with 10% (v v−1) foetal calf serum, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin. The fura-2-loading solution was based on PSS containing (mM): 140 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 7.7 glucose and 10 HEPES (pH adjusted to 7.2 with NaOH) to which pluronic-127 and fura-2 AM ester from a stock solution (1 mM fura-2/AM in DMSO) was added to obtain a final concentration of 0.02% pluronic-127 and 5 μM fura-2 AM. During experiments, rat intracardiac neurons were continuously superfused with ∼2 ml PSS/min. Racemic mixtures of thiopental, pentobarbital or ketamine were added to the extracellular solution, stirred thoroughly and bath applied prior to agonist application. Perfusion volume during application of the anaesthetics (<5 min) was approximately 10-fold the chamber volume (1 ml) to ensure that the desired anaesthetic concentrations were obtained before agonist application. Estimated clinically relevant anaesthetic concentrations were taken from the literature (Franks & Lieb, 1994; Krasowski & Harrison, 1999; Yamakura et al., 2001) and are free aqueous concentrations corrected for protein binding. Thiopental concentrations are nominal values not adjusted for the 9% sodium carbonate content of the powder. Agonists were applied to intracardiac neurons by pressure ejection (3–8 p.s.i.; Picospritzer II, General Valve Corp., Fairfield, NJ, U.S.A.) from a micropipette (3–5 μm diameter) positioned 50–100 μm from the cell soma. Nicotinic ACh receptor activation was obtained by focal application of maximally effective ACh concentrations (300–500 μM), with maximally effective concentrations of the muscarinic receptor antagonist atropine (100 nM) present both in the bath solution and extracellular pipette solution. In unclamped cells and perforated patch-clamp experiments, 500 μM ACh or 10 mM caffeine were applied for 1.2 s and a delay of at least 5 min between agonist applications was maintained. In dialysed patch-clamp experiments, 300 μM ACh was applied for 0.1 s and a delay of 70 s between ACh applications was maintained to minimize receptor desensitization. Agonists were applied several times before the superfusion of the anaesthetic and experiments were continued only if stable responses to agonist application were obtained. The pipette solution for perforated patch experiments contained (mM): 75 K2SO4, 55 KCl, 5 MgSO4 and 10 HEPES, titrated with N-methyl-D-glucamine to pH 7.2. A stock solution of 60 mg ml−1 amphotericin B in DMSO was prepared daily and diluted in pipette solution, providing a final concentration of 240 μg ml−1 amphotericin B in 0.4% DMSO, which was kept on ice and protected from light. The pipette solution for dialysed patch experiments contained (mM): 140 CsCl, 2 Mg2ATP, 5 Cs4BAPTA, 10 HEPES titrated with CsOH to pH 7.2 and that used to record Ca2+ channel currents was supplemented with 0.2 mM NaGTP to minimize current rundown. Whole-cell Ca2+ channel currents were recorded using Ba2+ as a charge carrier with the bath solution containing 140 TEACl, 4 BaCl2, 10 D-glucose and 10 HEPES-TEAOH (pH 7.2). All experiments were carried out at room temperature (22°C).
All chemical reagents used were of analytical grade. The following drugs were used: acetylcholine chloride, adenosine 5-triphosphate, amphotericin B, atropine sulphate, α-bungarotoxin, DMSO, (±) muscarine chloride, (Sigma Chemical Co., St Louis, MO, U.S.A.), ketamine hydrochloride (ICN Biomedicals Inc., Aurora, OH, U.S.A.), pentobarbitone sodium (Rhone Merieux Australia Ltd, Pinkenba, Queensland, Australia), thiopentone sodium (Jurox Pty. Ltd, Rutherford, New South Wales, Australia), caffeine (Fluka Chemie, Buchs, Switzerland), collagenase (type II; Worthington, Biochemical Corp., Lakewood, NJ, U.S.A.), BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) tetracesium salt, fura-2/AM, fura-2 pentapotassium salt and pluronic-127 (Molecular Probes, Eugene, OR, U.S.A.).
Peak (F340/F380) and resting (F340/F380) values of ratiometric fluorescence transients were determined by averaging F340/F380 values obtained from a 1.5 s period during the peak response and a 5 s period prior to the agonist application, respectively. Peak and resting F340/F380 ratios were then transformed to peak and resting [Ca2+]i, using Eq. (1), and peak increases in [Ca2+]i (Δ[Ca2+]i) were calculated by subtracting resting [Ca2+]i from peak [Ca2+]i. ACh-evoked peak current amplitudes and Δ[Ca2+]i in the presence of the anaesthetic were averaged and compared to the mean control response obtained in the absence of the anaesthetic in each individual cell. The mean control responses were determined by averaging responses obtained before and after superfusion with the anaesthetic to take into account cell rundown or incomplete recovery during washout that may occur during the experiment. Data are expressed as the mean±s.e.m. (n=number of cells) and were analysed statistically using Student's (paired, two-tailed) t-test with the level of significance being taken as P<0.05 (*), 0.01 (**) or 0.001 (***). Levels of significance were conservatively adjusted using the Bonferroni method when currents were tested for differences at several holding potentials.
In fura-2-loaded intracardiac neurons, the mean resting [Ca2+]i was 46±4 nM in the absence and 44±4 nM (n=30) in the presence of thiopental, pentobarbital and ketamine, respectively. The lack of effect of i.v. anaesthetics on resting Ca2+ levels indicates that, in unstimulated neurons, [Ca2+]i and thus transmembrane concentration gradients for Ca2+ ions were unchanged by the i.v. anaesthetics.
Clinically relevant concentrations of thiopental inhibit nAChR-mediated increases in [Ca2+]i in rat intracardiac neurons
In fura-2-loaded rat intracardiac neurons, focal application of 500 μM ACh to the cell soma in the presence of the muscarinic ACh receptor antagonist, atropine (100 nM) in the bath solution, evoked a rapid, transient increase in [Ca2+]i (Δ[Ca2+]i) of 70±5 nM above resting [Ca2+]i (n=21). The ACh-mediated transient increase in [Ca2+]i was not appreciably changed (<10%) in the presence of the 100 nMα-bungarotoxin, a selective antagonist of α7 nAChRs (Cuevas & Berg, 1998).
In the presence of the barbiturate, thiopental (25 μM=clinical EC50 (clinically relevant (half-maximal) effective concentration); Franks & Lieb, 1994; Krasowski & Harrison, 1999; Yamakura et al., 2001), however, the increase in [Ca2+]i evoked by focal application of 500 μM ACh in the presence of atropine was inhibited by 42% from 66±14 nM to 38±10 nM (P<0.01; n=6) as shown in Figure 1a. A concentration–response curve was obtained by applying ACh in the presence of atropine and varying concentrations of thiopental. Half-maximal inhibition of Δ[Ca2+]i by thiopental occurred at 28 μM, a value comparable to the clinical EC50 of thiopental, with a Hill coefficient of 1.24, as shown in Figure 1b.
Thiopental simultaneously inhibits nAChR-induced currents and increases in [Ca2+]i in voltage-clamped rat intracardiac neurons
To exclude any contribution of a secondary influx of Ca2+ ions to the increase in [Ca2+]i due to the involvement of voltage-dependent Ca2+ channels activated by nAChR-induced depolarization of the cell membrane, the influx of Ca2+ mediated by nAChR activation was studied under voltage-clamp conditions. Using the perforated patch recording configuration, fura-2-loaded neurons were held at −60 mV and application of 500 μM ACh evoked peak current amplitudes and increases in peak [Ca2+]i of −602±195 pA and 88±19 nM (n=4), respectively, in the presence of atropine. Bath application of 25 μM thiopental reduced the peak current amplitude by 61% to −237±84 pA (P<0.05; n=4) and the [Ca2+]i response by 53% to 41±10 nM (P<0.05) as shown in Figure 2. This result shows that a clinically relevant concentration of thiopental inhibits nAChR-induced peak current amplitude and simultaneous increases in [Ca2+]i, without any contribution of voltage-gated Ca2+ channels.
Further experiments were carried out using the dialysed patch recording configuration in which the pipette solution contained BAPTA to surmount any intracellular Ca2+ buffers. The ACh-evoked currents exhibited strong inward rectification and reversed close to 0 mV as described previously (Fieber & Adams, 1991; Adams & Nutter, 1992). As shown in Figure 3a and b, bath application of 25 μM thiopental reversibly inhibited nAChR-induced peak current amplitudes at negative holding potentials. Thiopental significantly inhibited peak current amplitudes at −120 mV by 38% from −1077±122 pA to −671±105 pA, at −80 mV by 38% from −732±81 pA to −454±70 pA and at −40 mV by 35% from −314±32 pA to −205±30 pA (n=11, P<0.0033 for each holding potential). A linear regression of the relative peak current amplitudes and the holding potentials revealed that the slope was not significantly different from zero (P<0.3), indicating that thiopental inhibition of nAChR-induced currents was voltage independent over the range of −40 to −120 mV (see Figure 3c). Similarly, the inhibition of nAChR-mediated peak current amplitude by 10 μM ketamine was relatively voltage independent at membrane potentials between −40 and −120 mV (n=3) consistent with that reported previously for nAChRs in PC12 cells (Furuya et al., 1999).
Clinically relevant concentrations of pentobarbital and ketamine also inhibit nAChR-mediated increases in [Ca2+]i in rat intracardiac neurons
Bath application of the barbiturate, pentobarbital (50 μM=clinical EC50; Franks & Lieb, 1994; Krasowski & Harrison, 1999; Yamakura et al., 2001), reversibly inhibited increases in [Ca2+]i evoked by focal application of 500 μM ACh by 40% from 77±7 to 46±11 nM (n=8; P<0.01) as shown in Figure 4a. Similarly, the dissociative anaesthetic, ketamine (10 μM=clinical EC50; Krasowski & Harrison, 1999; Yamakura et al., 2001), also reversibly inhibited ACh-induced increases in [Ca2+]i by 43% from 65±8 to 37±10 nM (n=7; P<0.05) as shown in Figure 4b. Bath application of pentobarbital and ketamine produced a concentration-dependent inhibition of the peak amplitude of ACh-induced increases in [Ca2+]i. A summary of the relative changes in ACh-induced increases in [Ca2+]i obtained in the presence of the i.v. anaesthetics is presented in Figure 4c.
Ca2+ release from caffeine-sensitive Ca2+ stores is not inhibited by clinically relevant concentrations of thiopental
Recently, we have shown that Ca2+ influx upon nAChR activation leads to CICR from intracellular, ryanodine-sensitive Ca2+ stores in rat intracardiac neurons (Beker et al., 2003). The secondary Ca2+ release from intracellular Ca2+ stores via RyR channels thus amplifies the nAChR-induced increase in [Ca2+]i. Given that thiopental has been suggested to inhibit ryanodine-induced Ca2+ release from the SR of papillary muscle cells (Komai & Rusy, 1994), the effect of thiopental on RyR channels was examined in rat intracardiac neurons. Caffeine, which is known to activate RyR channels while inhibiting IP3 receptor channels (Ehrlich et al., 1994), was used to examine the effects of thiopental on Ca2+ release from ryanodine-sensitive intracellular stores. As shown in Figure 5, application of 10 mM caffeine, which is likely to induce maximal Ca2+ release from ryanodine-sensitive stores, evoked a transient increase in [Ca2+]i of 92±14 nM (n=9), which was not significantly different from that obtained in the presence of 25 μM thiopental (90±14 nM; 98% of control, n=9; P<0.3). Increasing the duration of exposure to thiopental to 30 min had no appreciable effect on the amplitude of the caffeine-induced [Ca2+]i transient. These data indicate that the clinical EC50 of thiopental does not inhibit RyR-mediated Ca2+ transients in rat intracardiac neurons.
Intravenous anaesthetics at clinical EC50 do not inhibit depolarization-activated Ca2+ channel currents or muscarine- and ATP-evoked increases in [Ca2+]i in rat intracardiac neurons
In order to identify the primary molecular target(s) of the action of i.v. anaesthetics in rat intracardiac neurons, a series of experiments were carried out to evaluate the effect of i.v. anaesthetics on other cell-surface receptors and ion channels, which may mediate an increase in [Ca2+]i in these neurons. The activation of voltage-gated Ca2+ channels was examined using Ba2+ as the charge carrier and representative traces of Ba2+ currents evoked upon step depolarization from −100 to −20 mV in the absence (control) and presence of the i.v. anaesthetics are shown in Figure 6a. Peak Ba2+ current amplitude was not significantly changed during 10 min bath application of thiopental (25 μM, n=4), pentobarbital (50 μM, n=5) and ketamine (10 μM, n=7). However, raising the concentrations of either thiopental (100 μM) or pentobarbital (200 μM) four-fold reduced peak Ba2+ current amplitude by 19.6±3% (n=4) and 19.9±0.01% (n=3), respectively.
The activation of mAChRs and purinergic (P2X and P2Y) receptors have also been shown previously to elicit a transient increase in [Ca2+]i in rat intracardiac neurons (Liu et al., 2000; Beker et al., 2003). Bath application of thiopental (25 μM), pentobarbital (50 μM) and ketamine (10 μM) for up to 20 min duration, however, failed to inhibit increases in [Ca2+]i evoked by focal application of either 100 μM muscarine (Figure 6b) or 100 μM ATP (data not shown). Similarly, the transient increase in [Ca2+]i evoked upon activation of mAChRs by ACh in the presence of mecamylamine (10 μM) was unchanged in the presence of 25 μM thiopental (n=3). These data indicate that the mobilization of Ca2+ in rat intracardiac neurons via Ca2+ entry through voltage-gated Ca2+ channels and P2X receptor channels and release from intracellular IP3-sensitive Ca2+ stores (mAChRs and P2Y receptors) appears to be resistant to clinical EC50 of the i.v. anaesthetics.
The present study shows that activation of nAChR channels in fura-2-loaded rat intracardiac neurons induces an inward current and a transient increase in [Ca2+]i, which can be inhibited by clinically relevant concentrations of i.v. anaesthetics. Thiopental (25 μM) inhibited nAChR-induced increases in [Ca2+]i by 42% and a similar inhibition was observed with pentobarbital (50 μM; 40% inhibition) and with the dissociative anaesthetic, ketamine (10 μM; 43% inhibition). These results are consistent with previously reported studies using oocyte expression systems in which i.v. anaesthetics, including barbiturates and ketamine, have been shown to inhibit neuronal nAChR-mediated currents (see Krasowski & Harrison, 1999; Yamakura et al., 2001). On a molecular and cellular level, this corresponds with the clinical observation that no specific chemical group is required to produce the effects that occur during general anaesthesia (see Franks & Lieb, 1994). Resting [Ca2+]i was similar in the absence and the presence of the i.v. anaesthetics. This indicates that concentration gradients for Ca2+ were unaffected, and therefore changes in the driving force for Ca2+ cannot account for the inhibition of ACh-induced [Ca2+]i transients observed in the presence of the i.v. anaesthetics. Taken together, these results clearly show that clinically relevant concentrations of i.v. anaesthetics can modulate intracellular Ca2+ signals in response to nAChR activation and thus possibly modify various cellular, Ca2+-dependent functions, ranging from neuronal excitability to neurotransmitter secretion in intracardiac ganglia. Bath application of the i.v. anaesthetics prior to ACh application may simulate the situation occurring during the maintenance of general anaesthesia, whereby the receptor and anaesthetic are at equilibrium (Downie et al., 2000). The concentration–response curve for the thiopental inhibition of nAChR-induced increases in [Ca2+]i had a Hill coefficient of 1.24 and a half-maximal inhibition, IC50 of 28 μM, which is close to the clinical EC50 of thiopental, indicating that the nAChR may represent an important molecular target of clinically relevant concentrations of thiopental. Thus, the data presented strongly suggest that similar effects occur at intracardiac postganglionic neurons receiving nicotinic (vagal) stimulation in the presence of these i.v. anaesthetics during anaesthesia.
Under voltage-clamp conditions using the perforated patch recording configuration, ACh-induced transient increases in [Ca2+]i concomitant with rapid inward currents were inhibited in the presence of 25 μM thiopental. These data demonstrate that (i) thiopental inhibition of nAChR-induced Ca2+ transients in rat intracardiac neurons occurs without any contribution of voltage-gated Ca2+ channels and is similar to that observed in unclamped cells and (ii) a common mechanism underlies the simultaneous inhibition of nAChR-induced inward currents and [Ca2+]i transients by an i.v. anaesthetic, that is, is block of Ca2+ influx through nAChRs. Inhibition of ACh-evoked inward currents by thiopental has also been described in PC12 cells (Andoh et al., 1997) and for nAChRs exogenously expressed in Xenopus oocytes (Downie et al., 2000; Coates et al., 2001).
Under dialysed patch recording conditions, thiopental inhibited nAChR-mediated currents in rat intracardiac neurons in a voltage-independent manner at membrane potentials from −120 to −40 mV, consistent with that reported previously for thiopental inhibition of nAChRs in PC12 cells (Andoh et al., 1997) and human α7 homomeric nAChR channels expressed in Xenopus oocytes (Coates et al., 2001). The persistence of thiopental block of nAChR-mediated currents in dialysed patch experiments in which the pipette solution dictates the cytoplasmic composition indicates that the mechanism underlying the inhibition is not dependent on diffusible intracellular second messenger pathways. In contrast, the involvement of diffusible intracellular second messengers in mediating the inhibition of receptor channels by anaesthetics in Xenopus oocytes cannot be entirely eliminated (Downie et al., 2000). In particular, the pipette solution containing the rapid Ca2+ chelator, BAPTA, is likely to (i) surmount any endogenous intracellular Ca2+ buffer, (ii) to ensure a stable resting [Ca2+]i and (iii) to substantially suppress CICR. Therefore, it is unlikely that the inhibition of nAChR-induced currents observed in the presence of thiopental is due to a reduced driving force for Ca2+ caused either by reduced intracellular Ca2+ buffer capacities or increased CICR responses from internal stores. Furthermore, the voltage-independent block of nAChR-induced currents by ketamine is consistent with that reported previously for nAChRs in PC12 cells (Furuya et al., 1999). Thus, the inhibition of nAChR-mediated currents in rat intracardiac neurons by these anaesthetic agents is most likely due to a direct action on nAChR channels as proposed previously in studies using oocyte expression systems (Downie et al., 2000; Coates et al., 2001). Given that the proportion of the ACh-induced whole-cell current carried by Ca2+ through rat neuronal α7 nAChRs has been reported to be <10% (Fucile et al., 2003) and that α-bungarotoxin did not appreciably change nAChR-induced Ca2+ responses, then it is unlikely that i.v. anaesthetic inhibition of α7 homomers alone contributes significantly to the inhibition of the transient increase in [Ca2+]i observed in rat intracardiac neurons.
Given that CICR via the activation of RyRs has been shown to contribute to nAChR-induced [Ca2+]i transients in rat intracardiac neurons (Beker et al., 2003), the effects of thiopental on RyR-induced CICR were examined upon activation of RyRs with caffeine. Clinically relevant concentrations of thiopental had no effect on caffeine-induced increases in [Ca2+]i, indicating that thiopental is unlikely to inhibit CICR from internal stores by inhibiting RyR channels. It remains to be determined whether thiopental potentiation of Ca2+ release from ryanodine-sensitive Ca2+ stores may occur, as reported previously, for high concentrations of volatile anaesthetics on Ca2+ release from the SR in skeletal muscle (Kunst et al., 1999).
Further evidence for the selectivity of i.v. anaesthetic inhibition of nAChRs was obtained from the lack of effect of the i.v anaesthetics at clinically relevant concentrations on depolarization-activated Ba2+ currents and muscarine- and ATP-evoked [Ca2+]i transients. The voltage-dependent Ca2+ channels, which are predominantly N-type in rat intracardiac neurons (Xu & Adams, 1992), were unaffected by clinical EC50 of thiopental, pentobarbital and ketamine. Furthermore, the increase in [Ca2+]i that occurs upon the activation of either mAChRs or purinergic (P2X and P2Y) receptors is insensitive to the i.v. anaesthetics compared to nAChR-mediated [Ca2+]i transients in these neurons. Recombinant muscarinic receptor (M1–M3)-mediated [Ca2+] responses have been shown to be relatively insensitive to i.v. anaesthetics (Hirota et al., 2002) and the differential effects of thiopental on neuronal nAChRs and P2X receptors in PC12 cells have also been reported previously (Andoh et al., 1997).
In conclusion, we have shown that clinically relevant concentrations of i.v. anaesthetics modulate Ca2+ homeostasis in intracardiac neurons by inhibiting ACh-induced currents and [Ca2+]i transients. We suggest that the inhibition of ACh-induced [Ca2+]i transients by thiopental is due to a direct interaction with nAChR channels to inhibit Ca2+ influx and not a consequence of block of voltage-dependent Ca2+ channels, mAChRs, P2 purinoceptors or inhibition of Ca2+ release following RyR activation. Given that ACh is the primary neurotransmitter mediating parasympathetic (vagal) regulation of the heart and the presence of functional nAChR channels in intracardiac postganglionic neurons, clinically relevant concentrations of i.v. anaesthetics may modulate cardiac parameters during anaesthesia by binding to postsynaptic nAChRs and depressing ganglionic transmission. The observed reductions in ACh-induced current amplitude by the i.v. anaesthetics would also depress excitatory postsynaptic potential amplitude and therefore affect synaptic transmission in cardiac ganglia. A direct and selective effect of i.v. anaesthetics on nAChR channels in mammalian intrinsic cardiac neurons has not previously been taken into account when interpreting changes in heart rate and cardiac output during anaesthesia. Given the presence of presynaptic nAChRs in peripheral ganglia (see MacDermott et al., 1999), it will be of interest to determine if i.v. anaesthetics similarly target nAChRs in preganglionic parasympathetic nerve terminals.
We thank K. Bendeck, M. Both, Dr R.C. Hogg, Dr D. Uttenweiler, M. Vogel and F. von Wegner for critical discussion of this work. This work was supported by the Graduate School Biotechnology 388 of the German Research Foundation and a scholarship awarded to M. Weber by the German National Merit Foundation.