Propofol (2,6-diisopropylphenol) is widely used in clinical anesthesia and for sedation in the intensive care unit because of its rapid onset and clear emergence (1). Propofol, however, is often associated with adverse cardiovascular effects, including decreases in cardiac output (2) and arterial blood pressure (3). Propofol seems to have a greater depressant effect on the cardiovascular system than barbiturates (4). This depression may result from a decrease in sympathetic nerve activity (3), a reduction in baroreceptor control (5), and direct depression of myocardial contractility (4). However, the effect of propofol on the central nervous system (CNS), which is involved in the control of sympathetic nerve activity and cardiovascular function, has not been studied. It has been suggested that the hypothalamic paraventricular nucleus (PVN) neurons are involved in the regulation of the autonomic nervous and neuroendocrine systems and, in particular, in cardiovascular functions and body fluid balance (6,7). The PVN neurons of the hypothalamus are a heterogeneous structure comprised of neuronal populations that are grouped generally into magnocellular and parvocellular neurons (6). Previous electrophysiologic studies have revealed that both types of neurons in the PVN are controlled by a variety of neurotransmitters and modulators, including glutamate, γ-aminobutyric acid (GABA), norepinephrine, and neuropeptides (8–10). However, the effects of general anesthetics on these neurons have not been studied. GABA is the major inhibitory transmitter regulating PVN neurons, as well as other neurons, in the CNS (11). An immunohistochemical and electrophysiological study showed that the GABA neuron and its receptor are functionally expressed in the PVN (11). The GABAA receptor is a target of many general anesthetics, including volatile general anesthetics (12), barbiturates (13), and benzodiazepines (14), suggesting that propofol may affect PVN neurons.
The purpose of the current study was to explore the effects and mechanism of propofol on central neurons that are involved in the control of cardiovascular and sympathetic functions. For this purpose, we used a slice preparation of the PVN and evaluated the effect of propofol on the ionic currents of PVN neurons using the whole-cell mode of the patch-clamp technique.
The rats used in this study were treated and killed in accordance with national guidelines, and the protocol was approved by a local committee for animal care. The experimental procedure was broadly similar to that previously described (15). Male Wistar rats (days 15–21, postnatal) were anesthetized with isoflurane and then killed by decapitation. The brain was rapidly removed and placed in cooled (2°C–3°C) standard artificial cerebrospinal fluid (ACSF) containing: 140 mM of NaCl, 5.0 mM of KCl, 1.0 mM of MgCl2, 2.0 mM of CaCl2, 11.1 mM of glucose, and 10 mM of HEPES (pH value of 7.4 adjusted with NaOH), which was oxygenated with a 100% oxygen. The osmolarity was maintained at 290 mOsm with a pH value of 7.4. Coronal slices 200 μm in thickness, which included the PVN, were prepared using a vibrating brain slicer (DSK-2000; Dosaka, Kyoto, Japan) and allowed to equilibrate for ACSF for at least 20 min at 34°C and for up to 1 h at room temperature (24°C–26°C) before recording was started.
The electrodes were made with a puller (PB-7; Narishige, Tokyo, Japan) made from thick-wall borosilicate glass (GD-1.5; Narishige). Their resistance was 3–5 MΩ in the bath, with access resistances in the range of 8–12 MΩ. Slices were transferred into the recording chamber and continuously perfused (1.5–2.0 mL/min) with ACSF at room temperature (24°C–26°C). Whole-cell recordings were made from microscopically identified cells. Transmembrane currents were recorded using a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Foster City, CA) and stored on a hard disk via an analog/digital converter for later analysis. The signals were also displayed on a thermal rectigraph (Model 8M14; San-Ei, Tokyo, Japan). The stored data were analyzed using AxoGraph software (version 8.0; Axon Instruments).
The Cl− currents were recorded at a holding potential at −60 mV. The current-voltage relation of Cl− was measured using a ramp pulse of 200 ms from −80 to 20 mV. To block K+ currents, we used a pipette solution containing 110 mM of CsCl, 1 mM of MgCl2, 1 mM of CaCl2, 25 mM of TEA-Cl, 5 mM of 4-AP, 10 mM of EGTA, 10 mM of HEPES, and 4 mM of Na2-GTP and Mg-ATP (pH value of 7.2 adjusted with a Tris base). Furthermore, 100 μM of Cd, 100 μM of Ni, and 1 μM of tetrodotoxin (TTX) were added to the bath (HEPES-buffered solution) to block voltage-dependent Ca2+ channels and Na+ channels. In some experiments, Cs-methanesulfonate was used instead of CsCl.
Voltage-dependent Ca2+ currents were evoked by voltage steps from the holding potential of −80 mV to various depolarized test potentials (−60 to +20 mV). Leak currents and capacitive currents were canceled by off-line subtraction of Cd2+ (200 μM)-insensitive currents. The pipette solution used for Ca2+ current measurements contained 100 mM of TEA-Cl−, 5 mM of 4-AP, 1 mM of MgCl2, 1 mM of CaCl2, 10 mM of EGTA, 10 mM of HEPES, 0.3 mM of Na2-GTP, 4 mM of Mg-ATP, and 20 mM of creatine phosphate di-tris (pH value of 7.2 adjusted with a Tris base). The external solution was a HEPES-buffered solution with 1 μM of TTX and 100 μM of picrotoxin. The magnitude of Ca2+ current inhibition was evaluated as a percentage of the inhibition of the mean total currents that were measured just before and after the application of drugs.
Inhibitory postsynaptic currents (IPSCs) were recorded at a holding potential of −70 mV. IPSC were elicited by delivering a single stimulus (100-μs duration; 10–25 V) generated with an SS-102J isolator (Nihon Kohden, Tokyo, Japan) using bipolar stainless steel stimulating electrodes placed in a PVN neuron close to the recorded neuron. The external solution was a HEPES-buffered solution with 1 μM of TTX. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM) and DL-2-amino-5-phosphonovaleric acid (APV; 50 μM) were added to the extracellular solution to block inotropic glutamate receptors to study pharmacologically isolated GABAergic responses. Propofol (10−7–2 × 10−4 M) and other drugs were dissolved in HEPES-buffered solution and applied into the recording chamber by changing the perfusion line to the one that contained the drug.
Because we used a pipette solution that included a K+ blocker in this study, we could not classify electrophysiologically either a type 1 (putative magnocellular) or type 2 (putative parvocellular) neuron according to previously established criteria (16). All data are expressed as mean ± sd. The amplitude of Cl− currents in response to various concentrations of propofol was analyzed using one-way analysis of variance with the Sheffé F test. The inhibitory effects of strychnine and picrotoxin on propofol-induced currents were analyzed using a paired Student’s t-test. Relative Cl− current enhancement and inhibition of voltage-gated Ca2+ currents and the effects on IPSCs by propofol were analyzed using the Wilcoxon’s signed rank test. P < 0.05 was considered statistically significant. Propofol, TTX, strychnine, picrotoxin, CNQX, and APV were purchased from Sigma Chemical Company (St Louis, MO). We used 10% Intralipid (Pharmacia, Stockholm, Sweden) as the control.
A bath application of propofol (10−5–10−4 M) induced inward currents in PVN neurons at a holding potential of −60 mV (Fig. 1A). The averages of the amplitude of the inward currents induced by 10−5 M and 10−4 M of propofol were 334 ± 177 pA (n = 12) and 793 ± 211.6 pA (n = 12), respectively (Fig. 1B). To examine the ionic nature of the current induced by propofol, we determined the steady-state current-voltage relationships for PVN neurons. The reversal potential of the propofol-induced currents was 0.09 ± 2.24 mV (n = 14), which was close to the reversal potential of GABA-induced currents (0.71 ± 2.39 mV; n = 16) (Fig. 1C). The reversal potential of the propofol-induced currents was shifted to −38.45 ± 3.62 mV (n = 10) when methanesulfonate was used as a major anion in the pipette. The reversal potentials calculated from the Nernst equation using a Cl− concentration of a bath solution and a pipette solution for the CsCl and Cs-methanesulfonate experiments were −2.14 mV and −41.3mV, respectively. The good agreement in the two reversal potentials indicates that Cl− is responsible for the membrane current induced by propofol.
The next experiments were performed to determine the picrotoxin- and strychnine-sensitive components of the current induced by propofol. The current induced by 10−4 M of propofol was reversibly inhibited 87.7% ± 8.1% (n = 7) by 10−4 M of picrotoxin, a selective GABAA receptor antagonist, and 10.3% ± 6.2% (n = 7) by 10−6 M of strychnine, a selective glycine receptor antagonist (Fig. 2). Coapplication of 10−4 M of picrotoxin and 10−6 M of strychnine completely blocked the currents induced by 10−4 M of propofol, indicating that the propofol-induced Cl− current flows through the GABAA receptor-chloride ionophore complex and the glycine receptor-chloride ionophore complex. Intralipid did not induce Cl− currents in these tests.
To evaluate whether the facilitation of the GABA response by propofol was dependent on the agonist concentrations, propofol (10−6 M) was coadministered with variable concentrations of an agonist (GABA; 10−7–10−4 M). Propofol at 10−6 M, which induced little or no inward current, enhanced the GABA (10−6 M)-induced current (158.5% ± 16.8%) synergistically (Fig. 3), whereas propofol did not enhance currents induced by GABA (10−5 M and 10−4 M), which were near maximum in amplitude.
In the next series of experiments, the effects of propofol on the voltage-dependent Ca2+ currents were studied. Propofol induced an inhibition of voltage-gated Ca2+ currents elicited by a voltage step to −20 mV from a holding potential of −80 mV in a dose-dependent manner (Fig. 4, A and D). A bath application of propofol rapidly reduced the amplitude of voltage-gated Ca2+ currents, which recovered completely with the washout (Fig. 4B). The current-voltage relation of the propofol-induced inhibition of Ca2+ currents obtained by voltage steps from −60 to 20 mV revealed that high-voltage-activated currents were inhibited by propofol (Fig. 4C). The inhibition of Ca2+ currents induced by propofol was significant at concentrations of 10−5, 10−4, and 2 × 10−4 M (Fig. 4D). Intralipid had no effect on Ca2+ currents elicited by a voltage-step to −20 mV.
Because immunocytochemical and electrophysiological studies demonstrated that PVN neurons receive massive GABAergic synaptic connections (11), we evaluated the effects of propofol on IPSCs. Propofol (10−7-10−4 M) had no significant effects on the amplitude of the IPSCs (Fig. 5B), whereas it significantly enhanced the duration of IPSCs at concentrations of 10−5 M and 10−4 M (Fig. 5B). The decay time constant significantly increased to 119.4% ± 10.1% (n = 8) and 124.9% ± 14.4% (n = 8) of that of the control in response to 10−5 M and 10−4 M of propofol, respectively (Fig. 5B). Intralipid (phospholipid stabilized soybean oil) had little effect on the decay time constant (99.3% ± 12.3% of control; n = 4).
IV anesthetics cause various degrees of cardiovascular depression in vivo(2,3) and in vitro(17). The differences in cardiovascular depression could result from their differential effects on systemic vascular resistance (2), venous capacitance (18), the autonomic nervous system (3), and the heart (2,4). The present study provided the first evidence that propofol inhibits the activity of hypothalamic PVN neurons, which control cardiovascular and sympathetic functions at the central level. A bath application of propofol induced inward Cl− currents in a concentration-dependent manner (Fig. 1). Propofol has been reported to act on many kinds of receptors and ion channels in the CNS (19). However, little information is available about the effect of propofol on the central neurons, which control cardiovascular and sympathetic functions. The PVN neurons are comprised of neuronal populations that are grouped generally into magnocellular and parvocellular neurons, which can be either neuroendocrine or preautonomic neurons (6,7). Magnocellular neurons primarily synthesize arginine vasopressin (AVP) and oxytocin and secrete these hormones into the circulation from nerve terminals in the posterior pituitary (6). The preautonomic parvocellular neurons project to autonomic centers in the brainstem and spinal cord, which are involved in the regulation of heart rate, arterial blood pressure, and baroreceptor reflex (6). Both the electrical and the chemical stimulation of the PVN have been demonstrated to increase arterial blood pressure and renal sympathetic nerve activity in conscious rats (20). Thus, it is possible that the cardiovascular and sympathetic responses induced by propofol are, at least in part, mediated by the inhibitory actions of propofol on PVN neurons. The threshold concentration was less than 10−6 M, which is in accordance with the rat hippocampal pyramidal neurons (21). Shyr et al. (22) demonstrated that the brain level of propofol is roughly 10−6 M 40 min after propofol infusion at 60 mg · kg−1 · h−1 in rats. The data suggest that the concentration (10−6–10−4 M) of propofol used in our study is larger than a clinically applicable range for anesthesia. The difference of the effective dose between the clinical and cellular level may be due to comparing the clinical data and animal study. Because the Cl− currents induced by propofol were completely blocked by picrotoxin, a selective antagonist of the GABAA receptor, and strychnine, a selective antagonist of the glycine receptor, the currents seem to be mediated by GABAA and the glycine receptor in the PVN neurons. The strychnine-sensitive Cl− currents were 10.3% ± 6.2% of the total Cl− currents induced by propofol, a result that has been reported in dissociated rat hippocampal neurons (21) and spinal dorsal horn neurons (19).
Although 10−6 M of propofol did not induce many currents by itself, it synergistically enhanced GABA (10−6 M)-evoked currents. These results are consistent with a previous report indicating that propofol enhanced GABA-induced Cl− currents in rat hippocampal pyramidal neurons (21) and rat spinal dorsal horn neurons (19). The reason that the enhancement of GABA-mediated currents by propofol was observed in a narrow GABA-concentration range is that GABA, at the synaptic cleft, might reach a nearly maximum concentration around GABAA receptors in the postsynaptic membrane. This result may be supported by the lack of effects of propofol on the amplitude of IPSCs. Propofol at smaller concentrations may inhibit PVN neurons by enhancing the inhibitory effect of endogenously released GABA.
Next, we studied the effect of propofol on voltage-gated Ca2+ currents. A bath application of propofol produced rapidly reversible inhibition of Ca2+ currents in slice PVN neurons. Similar inhibition of Ca2+ currents by propofol has been reported in porcine tracheal smooth muscle cells (23) and rat cardiomyocytes (17). The inhibition was concentration-related, and the effective dose was 10−5 M or more. The functional significance of the modulation of the Ca2+ channel in PVN neurons has not been established; several lines of evidence suggest that the Ca2+ influx through the voltage-dependent Ca2+ channel during the action potential is important in somatodendritic AVP release (24). The inhibition of Ca2+ currents may suppress AVP release in PVN neurons. The suppression of AVP release may affect body fluid balance and the circulation system during surgical anesthesia. However, the inhibition of Ca2+ currents of preautonomic neurons in the PVN depresses the excitability of the neuron, which may induce cardiovascular depression. Although estimating effective concentrations in the brain and plasma in clinical situations of IV anesthetics is difficult because of their lipid solubility and interaction with plasma proteins, a previous study (25) has suggested that clinically relevant concentrations of propofol in such in vitro experiments would be approximately 10−6 M, which is close to the concentration of propofol that enhanced GABA-induced Cl− currents in PVN neurons in this study. A bath application of propofol produced reversible, concentration-related increases in the decay time constant of IPSCs, which is involved in the postsynaptic sites associated with GABAA receptors. Our results are in accordance with reports that volatile anesthetics, such as isoflurane, enflurane, and halothane (26), prolong the time course of GABAA receptor-mediated synaptic inhibition. Halothane has been reported to depress the amplitude of evoked IPSCs (26); however, the effect was slow; 15–20 min was required to reach steady-state depression. It is possible that the bath application time was too short to affect the amplitude of evoked IPSCs in our study. Although the precise determinants of the kinetics of the IPSCs are unknown, these results suggest that propofol enhanced the GABAA receptor-mediated synaptic transmission.
In conclusion, propofol at 10−6 M inhibited hypothalamic PVN neurons by enhancing inhibitory effects of endogenously released GABA through a postsynaptic mechanism. Furthermore, a larger concentration of propofol activated GABAA-receptor directly and inhibited the voltage-gated Ca2+ currents in the PVN neurons. These findings suggest that the cardiovascular and sympathetic depression caused by propofol are due, at least in part, to the inhibitory effects of propofol on PVN neurons, as directly measured by the whole-cell voltage-clamp method. These membrane alterations of the CNS probably interact with other direct action sites of propofol, including heart and blood vessels, leading to further cardiovascular depression.
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