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ANESTHETIC PHARMACOLOGY

The Effects of Isoflurane on Native and Chimeric Muscarinic Acetylcholine Receptors: The Role of Protein Kinase C

Sang-Hwan Do, MD, PhD^*, Ganesan L. Kamatchi, PhD^*, and Marcel E. Durieux, MD, PhD

*Department of Anesthesiology, University of Virginia Health Sciences Center, Charlottesville, Virginia; Department of Anesthesiology, Seoul National University College of Medicine, Seoul, Republic of Korea; and Department of Anesthesiology, University Hospital, Maastricht, The Netherlands

Address correspondence and reprint requests to Dr. Sang-Hwan Do, Department of Anesthesiology, University of Virginia, 1 Hospital Dr., PO Box 800710, Charlottesville, VA 22908-0710. Address e-mail to sd4z{at}hscmail.mcc.virginia.edu

	Abstract

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By using two electrode voltage clamps, we investigated the effects of isoflurane on m3 and chimeric m1/m3 muscarinic receptors and the role of protein kinase C (PKC) in the effects. Muscarinic receptors were expressed by injection of mRNA into Xenopus oocytes, and Ca²⁺-activated Cl^- currents were measured after the application of acetyl-ß-methylcholine. We constructed chimeric m1/m3 receptor DNA encoding the third intracellular loop of m1 and the remainder from the m3 receptor. Chimeric and m3 receptors were inhibited by isoflurane, but the m1 receptor was not. PKC activation with phorbol-12-myrisate-13-acetate (50 nM) decreased signaling of both chimeric and m3 receptors significantly. Chelerythrine (20 µM, PKC inhibitor) abolished the effect of isoflurane on chimeric and m3 signaling. Whereas isoflurane inhibition of chimeric and m3 receptors was completely reversible after washout with Tyrode’s solution for 3 min, treatment with okadaic acid (500 nM, protein phosphatase inhibitor) rendered the inhibition irreversible. Taken together, our results suggest that isoflurane inhibits m3 and chimeric m1/m3 muscarinic signaling by enhancing PKC activity and that the site of action is located outside of the third intracellular loop.

IMPLICATIONS: By use of the Xenopus oocyte expression system, we investigated the effects of isoflurane on muscarinic signaling and the role of protein kinase C in these effects. Our findings suggest that isoflurane inhibits muscarinic receptors through activation of protein kinase C and that the relevant phosphorylation sites are located outside the third intracellular loop.

	Introduction

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Inhaled anesthetics affect synaptic transmission in the central nervous system (CNS), and this effect is considered to form the basis for their general anesthetic actions (1,2). The mechanisms by which synaptic transmission is affected are multiple. A primary target is the -aminobutyric acid receptor (3,4), but other receptors and channels are also affected. Among these are the muscarinic acetylcholine receptors.

Aside from its circulatory and respiratory actions, muscarinic signaling plays an important role in the CNS (5). Brainstem muscarinic signaling modulates level of consciousness (6), and cortical muscarinic signaling affects memory and learning (7). Spinal muscarinic receptors, located primarily in the substantia gelatinosa of the dorsal horn, mediate antinociception (8). Many of these muscarinic effects are of obvious relevance to anesthesia.

Muscarinic receptors are members of the G-protein-coupled receptor superfamily. Five subtypes have been cloned since 1986. These five subtypes occur in two groups, the "odd" (m1, m3, m5) and the "even" (m2, m4), on the basis of sequence homology and second messenger signaling. The odd group couples to G_q or G_o proteins and signals primarily through increases in intracellular Ca²⁺; the even group couples to G_i proteins and signals through decreases in cyclic adenosine monophosphate production. Inhaled anesthetics affect muscarinic receptor function. Several anesthetics inhibit the binding of antagonist without affecting agonist binding (9) and alter the effect of guanine nucleotides on agonist binding (9). Halothane inhibits the m1 receptor signaling (10), and this inhibition depends on protein kinase C (PKC) activation by the anesthetic (11). However, it is not known whether this mechanism is specific to halothane and the m1 receptor or whether it can be extrapolated to other clinically used volatile anesthetics and other subtypes of muscarinic receptors. Isoflurane is of particular interest because it is often used clinically, and its effect on muscarinic signaling is quite different from that of halothane: isoflurane inhibits m3 signaling but has no effect on m1 signaling (12). Therefore, we studied the role of PKC in the inhibitory action of isoflurane on m3 receptor signaling.

To understand the effects of volatile anesthetics on muscarinic signaling, delineating the site of action is as important as elucidating the mechanism of action. The main difference between m1 and m3 receptors is the third intracellular (i3) loop. With the i3 loop and the amino and the carboxyl terminal excluded, the m3 sequence has 79% identity to the m1 sequence (13). Thus, we hypothesized that isoflurane’s action is mediated through this loop. To test this hypothesis, we constructed a chimeric m1/m3 muscarinic receptor DNA encoding the i3 loop from m1 and the remainder of the receptor structure from m3 (Fig. 1). By using this chimeric receptor, named m1/m3, we investigated the site of action of isoflurane on muscarinic receptor signaling.

View larger version (34K): [in this window] [in a new window]   Figure 1. A schematic diagram of the muscarinic signaling (A) and the muscarinic receptors (B) used in this study. (A) The double line indicates the cell membrane. Acetyl-ß-methylcholine (Mch) activates a muscarinic receptor in the membrane. The signal is transduced through a G protein (G) to phospholipase C (PLC). PLC generates inositoltrisphosphate (IP3) from phosphatidylinositolbisphosphate (PIP2). The IP3 releases Ca2+ from intracellular stores. Finally, this Ca2+ activates a Ca2+-dependent Cl- channel in the membrane, resulting in Cl- flux. (B) Numbers indicate amino acids. The middle segment shown in these receptors indicates the third intracellular (i3) loop. As indicated, the i3 loop of m3 receptor has 83 amino acids more as compared with the m1 receptor. The chimeric m1/m3 receptor is an m3 receptor that contains the i3 loop of the m1 receptor.

	Methods

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The study protocol was approved by the Animal Research Committee of the University of Virginia. Mature female Xenopus laevis frogs were purchased from Xenopus I (Ann Arbor, MI) and fed regular frog brittle twice weekly. For removal of oocytes, frogs were anesthetized in 500 mL of 0.2% 3-aminobenzoic acid ethyl ester (Sigma, St. Louis, MO) in water until they were unresponsive to painful stimuli (toe pinching), and they were operated on ice. A 5-mm-long incision was made in the lower lateral abdominal quadrant, and a lobule of ovarian tissue containing approximately 200 oocytes was removed and placed immediately in modified Barth’s solution [containing, in mM, NaCl 88, KCl 1, NaHCO₃ 2.4, CaCl₂ 0.41, MgSO₄ 0.82, Ca(NO₃)₂ 0.3, gentamicin 0.1, and HEPES 15, with the pH adjusted to 7.6]. The oocytes were defolliculated with gentle shaking for approximately 2 h in calcium-free OR-2 solution (containing, in mM, NaCl 82.5, KCl 2, MgCl₂ 1, HEPES 5, and collagenase type Ia 0.1%, with the pH adjusted to 7.5) and then incubated in modified Barth’s solution at 18°C.

The rat m1 and m3 acetylcholine receptor complementary DNAs (cDNAs) in a commercial vector (pGEM1; Promega, Madison, WI) were obtained from Dr. T. I. Bonner (National Institute of Mental Health, Bethesda, MD). Chimeric m1/m3 cDNA was constructed with routine molecular biology techniques. Briefly, all cDNA fragments necessary for the construction of the chimeric cDNA were obtained from polymerase chain reactions by use of m1 or m3 receptor cDNA as templates. These polymerase chain reaction products carrying restriction enzyme sites at their 5' and 3' ends were gel-purified and digested with the respective restriction enzymes. The matching fragments of cDNA for the chimera were subcloned in pcDNA 3.1 (Invitrogen, Carlsbad, CA) by transforming DH5 cells (Gibco BRL, Gaithersburg, MD) with the protocols recommended by the suppliers. The transformant with the proper cDNA insert was sequenced (Core Facility, University of Virginia), and its homology with the parent cDNA was confirmed. This plasmid DNA was linearized, and messenger RNA (mRNA) was synthesized in vitro with a commercially available kit (Ambion, Austin, TX). The resulting mRNA was quantified spectrophotometrically and diluted in sterile RNase-free water. This mRNA was used for the cytoplasmic injection of oocytes in a concentration of 5 ng/30 nL by using an automated microinjector (Drummond "Nanoject"; Drummond Scientific Co., Broomall, PA). Injected oocytes were incubated for 72 h at 18°C in modified Barth’s solution before study.

Experiments were performed at room temperature (approximately 22°C). A single defolliculated cell was placed in a recording chamber (0.5-mL volume) and perfused with 3 mL/min Tyrode’s solution (containing, in mM, NaCl 150, KCl 5, CaCl₂ 2, MgSO₄ 1, dextrose 10, and HEPES 10, with the pH adjusted to 7.4). Microelectrodes were pulled in one stage from capillary glass (10-µL tube; Drummond Scientific, Broomall, PA) on a micropipette puller (model 700C; David Kopf Instruments, Tujunga, CA). Tips were broken to a diameter of approximately 10 µm, providing a resistance of 1–3 M, and were filled with 3 M KCl. The oocytes were voltage-clamped by using a two-microelectrode oocyte voltage clamp amplifier (OC725A; Warner Corporation, New Haven, CT) connected to a data acquisition and analysis system running on a personal computer. The acquisition system consisted of a DAS-8A/D conversion board (Keithley-Metrabyte, Taunton, MA), and analysis was performed with OoClamp software (10). All measurements were performed at a holding potential of -70 mV. Cells that did not show a stable holding current of <1 µA (<5% of cells tested) were excluded from analysis. Ca²⁺-activated Cl^- currents (I_Cl(Ca)) were sampled at 125 Hz and recorded for 5 s before and for 55 s after the start of application of the agonist (acetyl-ß-methylcholine; Mch) delivered at appropriate 50% effective concentration (EC₅₀) values. Responses were quantified by determination of peak current, measured in microamperes. Each experiment was performed with oocytes from at least three different frogs (three to five oocytes from each frog).

Mch was diluted in Tyrode’s solution and perfused over the oocyte for 20 s (3 mL/min). To determine the effect of isoflurane, output from an isoflurane vaporizer (1.25% and 2.5%, approximately 1 and 2 minimum alveolar anesthetic concentration [MAC], respectively) was bubbled through a reservoir filled with 40 mL of Tyrode’s solution. Air at a flow rate of 500 mL/min was used as the carrier gas, and at least 10 min was allowed for equilibration. We have previously shown this technique to result in stable bath concentrations of approximately 0.70 mM (14). To study the effect of PKC activation on muscarinic signaling, phorbol-12-myristate-13-acetate (50 nM) in Tyrode’s solution was perfused for 5 min before the application of Mch. To determine the effect of isoflurane after PKC inhibition, oocytes were incubated in the PKC inhibitor chelerythrine (20 µM) for 1 h, and then Mch was applied with or without isoflurane (3 min). To investigate the ability of protein phosphatase inhibition to "lock in" the effect of isoflurane, oocytes were incubated with okadaic acid (500 nM) for 1 h. Okadaic acid-treated oocytes were exposed to isoflurane (2 min), which was then washed out with Tyrode’s solution (3 min) before measurements of Mch-induced I_Cl(Ca).

Molecular biology reagents were obtained from Promega. Other chemicals were obtained from Sigma. Isoflurane was purchased from Ohmeda (Liberty Corner, NJ).

Results are reported as mean ± SEM. Statistical analyses were performed by using either t-tests or one-way analysis of variance. P < 0.05 was considered significant.

	Results

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Whereas uninjected oocytes were unresponsive to Mch (data not shown), oocytes injected with m1 or m3 receptor mRNA responded to application of Mch with a transient inward current (Fig. 2A), as described previously (12). Oocytes injected with m1/m3 receptor mRNA responded in a similar way (Fig. 2A). The response was concentration dependent, and Mch EC₅₀ values were determined from the Hill equation to be 170 nM for m1, 16 nM for m3, and 17 nM for m1/m3 (GL Kamatchi, written communication, June 1, 2000). Thus, pharmacologically, the m1/m3 receptor behaves like the parent m3 receptor. In the remaining studies, we used 200 nM Mch to activate m1 receptors and 20 nM Mch to activate m3 or m1/m3 receptors.

View larger version (10K): [in this window] [in a new window]   Figure 2. Calcium-dependent chloride current (ICl(Ca)) induced by acetyl-ß-methylcholine (Mch) in the presence or absence of isoflurane (2 minimum alveolar anesthetic concentration). The currents showed increased responses with the increase in the concentration of Mch (A). Isoflurane inhibited m3 and m1/m3 muscarinic responses significantly. Isoflurane had no effect on Mch-induced ICl(Ca) in oocytes expressing m1 receptors (B). The horizontal bar indicates the duration of Mch application.

PKC activation by brief treatment with phorbol-12-myristate-13-acetate (50 nM) decreased m3 signaling significantly (from 0.4 ± 0.1 µA to 0.1 ± 0.04 µA, 75% decrease, P < 0.001, Fig. 3). Similarly, PKC activation decreased m1/m3 responses (from 1.4 ± 0.3 µA to 0.4 ± 0.1 µA, 71% decrease, P < 0.05, Fig. 3). These findings suggest that isoflurane may inhibit m3 and m1/m3 signaling by activating PKC. To test this hypothesis, we determined the effect of isoflurane on m3 and m1/m3 signaling in the presence of the PKC inhibitor chelerythrine (20 µM). Chelerythrine itself induced no significant changes in muscarinic signaling: 0.4 ± 0.1 µA (control) versus 0.6 ± 0.1 µA (chelerythrine) on m3 receptors (n = 12 in each group) and 1.1 ± 0.2 µA (control) versus 1.4 ± 0.2 µA (chelerythrine) on m1/m3 receptors (n = 20 in each group). However, when chelerythrine-treated cells were exposed to isoflurane, responses to Mch were no longer inhibited (Fig. 4). Thus, PKC inhibition blocks the action of isoflurane. The similarity of effect on m3 and m1/m3 receptors suggests that the PKC phosphorylation sites involved in this process are located outside the i3 loop.

View larger version (12K): [in this window] [in a new window]   Figure 3. Effects of protein kinase C (PKC) activation on m1/m3 or m3 signaling. After perfusion with phorbol-12-myristate-13-acetate (PMA) (PKC activator; 50 nM) for 5 min, oocytes injected with m3 or m1/m3 messenger RNA showed a significant decrease in acetyl-ß-methylcholine (Mch)-induced current. *P < 0.05, **P < 0.001 compared with control. Data are mean ± SEM; n = 12–13.

View larger version (26K): [in this window] [in a new window]   Figure 4. Effects of protein kinase C (PKC) inhibition on m1/m3 or m3 signaling; 1 or 2 minimum alveolar anesthetic concentration (MAC) isoflurane (3 min) significantly decreased the calcium-dependent chloride current (ICl(Ca)) induced by Mch on both m1/m3 and m3 receptors. However, oocytes incubated with chelerythrine (PKC inhibitor) for 1 h did not show significant changes in current after exposure to isoflurane. *P < 0.05 compared with control. Data are mean ± SEM; n = 15–19. Mch = acetyl-ß-methylcholine.

View larger version (31K): [in this window] [in a new window]   Figure 5. Effects of protein phosphatase inhibition on m1/m3 or m3 signaling after exposure to isoflurane and subsequent washout. Inhibitory effects of isoflurane on chimeric or m3 signaling were completely reversible after washout with Tyrode’s solution (3 min). However, oocytes incubated with okadaic acid (protein phosphatase inhibitor) for 1 h showed a significant decrease in Mch-induced calcium-dependent chloride current (ICl(Ca)) after exposure to isoflurane and subsequent washout. *P < 0.05 compared with control, #P < 0.05 compared with the Isoflurane + Washout group. Data are mean ± SEM; n = 12–15. Mch = acetyl-ß-methylcholine; MAC = minimum alveolar anesthetic concentration.

	Discussion

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The effect of isoflurane was modified in a similar manner in m3 and m1/m3 receptors by PKC inhibition and phosphatase inhibition, again suggesting a target site outside the i3 loop. PKC inhibition with chelerythrine abolished the depressant effect of isoflurane in both m3 and m1/m3 signaling. Protein phosphatase inhibition with okadaic acid "locked in" the depressant effect of isoflurane in both receptor constructs. Taken together, m3 muscarinic signaling inhibition by isoflurane seems to be mediated by PKC activation, and the relevant phosphorylation sites seem to be located outside the i3 loop. In a study with m1 muscarinic receptors, PKC primarily phosphorylated sites outside of the i3 loop and in the carboxyl-terminal tail of the receptor (15).

Several potential limitations should be considered when interpreting our findings. Xenopus oocytes are clearly different from native human cells. However, this model has been used frequently for the study of muscarinic receptors by us (10,12) and others (11,16), and it is well established that receptors function similarly in oocytes as in native human cells. Our experiments were performed at room temperature, whereas the receptor was from a homeothermic animal. However, there is no evidence that such temperature difference results in significant changes in kinetics and function for these receptor types. Furthermore, we believed that it was more important to keep the cell membrane at its normal temperature, because an abnormally high temperature could cause changes in lipid bilayer structure and receptor functioning. Finally, although chimeric constructs may be useful in delineating sites of action, it should be realized that improper folding of the mutated amino acid chain may cause an effect at a distance to be misinterpreted as an effect at the changed site.

Considerable evidence suggests an important role for PKC in the regulation of G-protein-coupled receptors (17). PKC is activated by intracellular Ca²⁺ and diacylglycerol, which are produced during activation of G-protein-coupled signaling. Phosphorylation causes desensitization of receptor function (18). Therefore, phosphorylation by activation of PKC can be regarded as a process of negative feedback to regulate G-protein-coupled receptor function. Anesthetics might inhibit receptor function by directly activating this feedback loop. An alternative explanation is that isoflurane enhances protein phosphorylation by inhibiting protein phosphatases. However, phosphatase inhibition itself did not affect signaling significantly. Our findings with m3 and isoflurane are very similar to those obtained by Minami et al. (11) regarding the effects of halothane and alcohols on m1 signaling. In addition, they showed that mutation of a PKC consensus site (outside the i3 loop) in the metabotropic glutamate receptor abolished the inhibitory effect of halothane on those receptors (19). Thus, inhibition by PKC activation may be a common mechanism for anesthetic actions on G-protein-coupled receptors. The effects of volatile anesthetics on PKC have been variably reported (20).¹ In contrast to evidence that halothane activates PKC, there are few reports regarding isoflurane. Isoflurane may enhance PKC-mediated vasoconstriction (21).

In conclusion, our results suggest that isoflurane inhibits m3 muscarinic signaling by enhancing PKC activity. The site of action is located outside of the i3 loop. These data, together with other materials in the literature, suggest that this mechanism may explain the interactions of several anesthetics with G-protein-coupled receptors.

	Footnotes

 
Presented in part at the 2001 IARS Clinical and Scientific Congress, Ft. Lauderdale, FL, March 17, 2001.

¹ Firestone S, Gray K, Firestone LL. General anesthetics and ethanol inhibit enzymatic activity of CNS-derived protein kinase C [abstract]. Anesthesiology 1990;73:A706.

	References

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999