Midazolam Induces Expression of c-Fos and EGR-1 by a... : Anesthesia & Analgesia (original) (raw)
Many of the clinically used IV anesthetics are thought to produce their pharmacologic effects through their interaction with specific receptor molecules in the central nervous system (CNS). Barbiturates, benzodiazepines, and propofol bind with the subunit of the γ-aminobutyric acid (GABA)A receptor and facilitate its function (1). On the other hand, the main site of action of ketamine is the _N_-methyl-d-aspartate receptor channel (2). Functions of these ion channels are modulated by binding of these IV anesthetics, and recovered immediately after washout of the anesthetics.
In contrast to functional changes of cells acutely elicited by modulation of ion channel functions by IV anesthetics, long-term changes in cellular functions lasting even after the elimination of the drugs have not been noticed. However, anesthesiologists should be aware of anesthesia-related long-term changes in neuronal functions, because these changes might result in tolerance and dependence on hypnotic drugs, and might produce subtle changes in neural functions after anesthesia. A possible mechanism for drug-induced long-term modification of cellular functions is based on gene expression changes induced by the drugs. We have previously reported that opioids can lead to activation of extracellular signal-regulated kinases (ERKs) (3), a class of the mitogen-activated protein kinase (MAPK), which can activate various transcription factors leading to changes in gene expression (4). Furthermore, it has been reported that local anesthetics inhibit the depolarization-induced or muscarinic receptor-mediated MAPK activation in a neuronal cell line (5). This finding may imply that local anesthetics can modulate gene expression in neuronal cells. Thus, it has been demonstrated that some of the drugs used in clinical anesthesia affect the gene expression in neuronal cells, which might lead to long-term changes in neural functions.
In the present investigation, we tested whether clinically used IV anesthetics, including thiopental, ketamine, propofol, midazolam, and diazepam induce changes in gene expression in PC12 rat pheochromocytoma cells. The results show that midazolam induces expression of transcription factors, c-Fos and EGR-1, and that the induction is mediated by ERKs. This finding may suggest that midazolam can affect expression of a variety of genes by the induction of c-Fos and EGR-1.
Methods
Dulbecco’s modified Eagle’s medium was purchased from GIBCO (Grand Island, NY). Fetal bovine serum and horse serum were from ICN Biomedicals (Aurora, OH). Horseradish peroxidase-conjugated antirabbit immunoglobulin and an enhanced chemiluminescence detection system were obtained from Amersham Pharmacia Biotechnology (Uppsala, Sweden). PD98059 and PK11195 were from New England Biolabs (Beverly, MA) and Research Biochemicals (Natick, MA), respectively. Midazolam and flumazenil were from Yamanouchi (Tokyo, Japan). Thiopental, ketamine, propofol, and diazepam were obtained from Tanabe (Osaka, Japan), Sankyo (Tokyo, Japan), AstraZeneca (Osaka, Japan), and Takeda (Osaka, Japan), respectively. All the other reagents were obtained from Wako (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), and Sigma (St. Louis, MO).
PC12 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 5% horse serum, 50 μg/mL streptomycin, and 50 U/mL penicillin at 37°C, in the humidified atmosphere of 95% air and 5% carbon dioxide. For experiments, PC12 cells were grown to subconfluence in 6-well culture plates (Becton Dickinson, Franklin Lakes, NJ), and serum-starved for 8 h. After serum starvation, cells with or without pretreatment were treated with IV anesthetics or GABAA receptor agonists, GABA and muscimol, of the indicated concentrations at 37°C for the indicated times. In the experiments testing whether these drugs can induce c-Fos and EGR-1 expression, we treated the cells with 100 μM of these drugs, which are not equipotent to each other but can be considered as a sufficiently large concentration. In the experiments testing the effects of pretreatment with inhibitors or antagonists, inhibitors or antagonists were also present during treatment of the cells with IV anesthetics.
After stimulation, cells were washed twice with ice-cold phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4), and homogenized in 200 μL of ice-cold cell lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2.5 mM EDTA, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate, 1% deoxycholic acid, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mg/mL leupeptin. The supernatant obtained by centrifugation of the homogenate at 20,000 g for 5 min was subjected to immunoblot analysis. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis, and transferred electrophoretically to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). Blocking of the membrane was performed for 1 h in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) (20 mM Tris [pH 7.5], 137 mM NaCl, 0.1% Tween-20) supplemented with 10% nonfat dry milk. After blocking, the membrane was incubated for 2 h at room temperature with antibodies. Anti-c-Fos antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-EGR-1 antibody (Santa Cruz Biotechnology) diluted 1:2000 in TBS-T supplemented with 5% nonfat dry milk were used to detect c-Fos and EGR-1, respectively. Phospho-specific p44/42 MAPK antibody (New England Biolabs) diluted 1:1000 in TBS-T supplemented with 5% bovine serum albumin was used to detect phosphorylated ERKs. Then the membrane was incubated for 1 h at room temperature with horseradish peroxidase-conjugated antirabbit immunoglobulin. After washing with TBS-T, the protein-antibody complex was visualized using the enhanced chemiluminescence detection system.
Immunoblots were analyzed by using NIH Image version 1.6 software (National Institutes of Health, Bethesda, MD). The results are expressed as mean ± sd. Statistical analyses of data were performed by using analysis of variance followed by the Fisher’s protected least significance difference test. P values < 0.05 were considered statistically significant.
Results
First, we tested whether clinically used IV anesthetics induce changes in c-Fos and EGR-1 expression. It has been reported that expression of these immediate early gene products is induced by a variety of stimuli including depolarization and neurotransmitters in PC12 cells (6,7). Furthermore, these proteins, which function as transcription factors, affect expression of other genes, and can be involved in long-term modulation of neural functions by anesthetic drugs. Figure 1 shows that incubation of PC12 cells with 100 μM midazolam for 60 min induces significant c-Fos and EGR-1 expression. In contrast, 100 μM propofol, 100 μM ketamine, 100 μM thiopental, and 100 μM diazepam did not significantly affect the expression level of c-Fos and EGR-1. Figure 2A demonstrates that c-Fos and EGR-1 expression peaked at approximately 60 min after the addition of 100 μM midazolam, and declined gradually thereafter. As shown in Figure 2B, midazolam-induced expression of c-Fos and EGR-1 was significantly observed at a concentration not <100 μM.
Effects of IV anesthetics on the expression of c-Fos and EGR-1. PC12 cells were treated with vehicle (lane 1), thiopental (lane 2), ketamine (lane 3), propofol (lane 4), midazolam (lane 5), or diazepam (lane 6) (100 μM each) for 60 min at 37°C. The expression of c-Fos and EGR-1 was assessed by using immunoblot analysis. A typical result from three independent experiments is shown. The positions of c-Fos and EGR-1 are indicated.
Midazolam-induced c-Fos and EGR-1 expression in PC12 cells. The expression of c-Fos and EGR-1 was assessed by using immunoblot analysis and densitometry. *c-Fos expression different from control at P < 0.05. #EGR-1 expression different from control at P < 0.05. (A) Time dependence of the midazolam (100 μM)- induced c-Fos and EGR-1 expression. Data are mean ± sd from four independent experiments. (B) Dose dependence of the c-Fos and EGR-1 expression induced by incubation with midazolam for 60 min. Data are mean ± sd from five independent experiments.
We next tested the involvement of the GABAA receptor in the induction of c-Fos and EGR-1 expression by midazolam. Figure 3A shows that application of agonists for the GABAA receptor, GABA (100 μM) and muscimol (100 μM), do not significantly induce c-Fos or EGR-1 expression. Furthermore, as shown in Figure 3A, neither flumazenil, an antagonist for the benzodiazepine receptor in the CNS, nor PK11195, an antagonist for the peripheral benzodiazepine receptor, affected the midazolam-induced c-Fos and EGR-1 expression (Fig. 3A).
Effects of pretreatment on the midazolam-induced c-Fos and EGR-1 expression in PC12 cells. The expression of c-Fos and EGR-1 was assessed by using immunoblot analysis and densitometry. *c-Fos expression different from control at P < 0.05. #EGR-1 expression different from control at P < 0.05. (A) PC12 cells with or without pretreatment with 10 μM flumazenil or 10 μM PK11195 for 60 min were stimulated with γ-aminobutyric acid (GABA), muscimol, or midazolam (100 μM each) for 60 min. Data are mean ± sd from three independent experiments. (B) PC12 cells with or without pretreatment with 50 μM PD98059 for 60 min were stimulated with 100 μM midazolam for 60 min. Data are mean ± sd from five independent experiments.
To test the involvement of ERK in midazolam-induced c-Fos and EGR-1 expression, we used PD98059, an inhibitor for MAPK/ERK kinase, a kinase that activates ERKs by phosphorylation. Pretreatment of PC12 cells with 50 μM PD98059 abolished the midazolam-induced c-Fos and EGR-1 expression (Fig. 3B), suggesting the involvement of ERKs in the midazolam effect.
Finally, we examined whether ERKs are indeed activated by the application of midazolam to PC12 cells. Activation of ERKs was assessed by immunoblot analysis using an antibody specific for ERKs phosphorylated at the amino acid residues threonine 202 and tyrosine 204 (8). Figure 4 demonstrates that the amount of phosphorylated ERKs (ERK1 and ERK2) is increased immediately after the addition of midazolam, peaked at 10 min, and declined gradually to the control level by 60 min.
Midazolam-induced activation of extracellular signal-regulated kinases (ERKs) in PC12 cells. PC12 cells were stimulated with 100 μM midazolam for the indicated times. Phosphorylated ERKs were detected by using immunoblot analysis. (A) A typical result from three independent experiments is shown. Positions of phosphorylated ERKs (p-ERK1 and p-ERK2) are indicated. (B) Densitometric analysis of time-dependent phosphorylation of ERKs induced by midazolam. The amount of phosphorylated ERKs (p-ERK1 and p-ERK2) is expressed as ratio to control. Data are mean ± sd from three independent experiments. *Different from control at P < 0.05.
Discussion
In this investigation, we tested the induction of immediate early gene products c-Fos and EGR-1 that are involved in transcription of a variety of genes, by IV anesthetics. We showed that midazolam time- and dose-dependently induces expression of c-Fos and EGR-1, through the mechanism mediated by ERKs in the PC12 rat pheochromocytoma cell line.
The PC12 cell line has been widely used as a model of neuronal cells. In the field of anesthesiology, this cell line has been used to investigate the effects of anesthetics on cellular signal transduction mechanisms. For example, Kansha et al. (5) reported that dibucaine and tetracaine inhibit the MAPK activation and c-Fos expression mediated by L-type Ca2+ channels in PC12 cells.
Benzodiazepines, including midazolam, bind with the central benzodiazepine receptor—a part of the GABAA receptor located in the CNS—and produce a facilitatory effect on the GABAA receptor channel, resulting in their sedative, hypnotic, and anxiolytic effects (1). Although reverse transcriptase polymerase chain reaction detected β3, γ2, and δ subunits of the GABAA receptor in PC12 cells (9), no GABA-evoked current was recorded by the patch clamp technique (10), suggesting the absence of functional GABAA receptor channels in PC12 cells. In the present study, we demonstrate that the expression of c-Fos and EGR-1 is not significantly affected by flumazenil, an antagonist for the central benzodiazepine receptors. Furthermore, GABAA receptor agonists, GABA and muscimol, did not induce expression of c-Fos and EGR-1. These results suggest that midazolam activates a mechanism distinct from the GABAA receptor, leading to expression of c-Fos and EGR-1.
Benzodiazepines also bind with other receptors, known as the peripheral benzodiazepine receptors, located mainly in peripheral tissues and glial cells in the brain (11). A previous study showed that PC12 cells have high-affinity peripheral benzodiaze-pine receptors (12). Furthermore, it was shown that peripheral-type benzodiazepine derivatives do not significantly affect the c-Fos expression by themselves, but rather facilitate nerve growth factor-induced expression of c-Fos in PC12 cells (13). However, we demonstrated that the midazolam-induced c-Fos and EGR-1 expression was not inhibited by PK11195, an antagonist for the peripheral benzodiazepine receptor, indicating that this response is not mediated by the peripheral benzodiazepine receptor in the PC12 cells.
Yamakage et al. (14) demonstrated that diazepam and midazolam have inhibitory effects on the activity of voltage-dependent Ca2+ channels in canine tracheal smooth muscle cells with 50% inhibitory concentration values of 1–10 μM, which was not affected by flumazenil and PK11195. Moreover, it was reported that voltage-dependent Na+ and K+ currents were reduced by 10–100 μM midazolam and diazepam by a non-GABAergic mechanism in NG108-15 neuroblastoma-glioma hybrid cells (15). Thus, it is conceivable that benzodiazepines can interact with molecules other than the central and peripheral benzodiazepine receptors to produce biological effects.
Our data demonstrate that PD98059, an MAPK/ERK kinase inhibitor, almost completely inhibited the midazolam-induced c-Fos and EGR-1 expression, suggesting the involvement of ERKs in the response to midazolam. This result is compatible with the previous report showing the attenuation of growth hormone-induced c-Fos and EGR-1 expression by PD98059, which suggested the involvement of ERKs in the induction of these immediate early gene products by stimuli from outside of cells (16). It has been shown that ERKs phosphorylate the transcription factor Elk-1, and that phosphorylated Elk-1 bound with serum response factors interacts with the serum response element and facilitates transcription of the c-fos gene (4). ERKs can be activated by several signal transduction mechanisms, including G-protein-coupled receptors, growth factor receptors, protein kinase C and Ca2+ influx. However, it has not been known whether midazolam can activate these mechanisms without involvement of the GABAA receptor. We should attempt to elucidate the molecular mechanism involved in midazolam-induced ERK activation.
c-Fos can form heterodimers with transcription factors of the Jun family, such as c-Jun, Jun-B, and Jun-D, bind with the AP-1 site, and affect the transcription of target genes (17). Members of the EGR family, EGR-1–4, induced by growth factors and synaptic activity, bind with the EGR response element and regulate the transcription of the target genes (18). Therefore, our results showing the midazolam-induced c-Fos and EGR-1 expression may imply that the administration of midazolam can induce changes in gene expression and long-term effects in the CNS. As a next step of investigation, it will be interesting to examine which genes are activated by c-Fos and EGR-1 induced by midazolam in neuronal cultured cells and in the CNS. It is possible that proenkephalin and prodynorphin genes are activated by midazolam, because these genes are controlled by c-Fos (19,20).
ERKs also affect signal transduction mechanisms other than those related to gene expression (8). Cytosolic phospholipase A2 is phosphorylated and activated by ERKs (21). Therefore, it may be conceivable that midazolam induces cytosolic phospholipase A2 activation and an increase in production of prostaglandins and leukotrienes in neuronal cells, resulting in modulation of cellular functions and synaptic functions.
In the present study, the midazolam-induced immediate early gene expression was significant at concentrations not <100 μM. This phenomenon may not be relevant when the usual dose of midazolam is systemically administered, because plasma concentrations of benzodiazepines used clinically are approximately between 0.1 and 50 μM (22). However, when large micromolar local concentrations are achieved after intrathecal (23) or caudal (24) injection of midazolam for analgesia, immediate early gene expression might be induced in the spinal cord neurons. Whether c-Fos and EGR-1 are induced in neuronal cells by midazolam administered via a variety of routes should be tested by conducting animal experiments.
In conclusion, midazolam induces c-Fos and EGR-1 expression in PC12 cells through a non-GABAergic mechanism involving ERKs. It remains to be investigated whether similar induction of gene expression by midazolam can be observed in vivo.
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