Anesth Analg 2007;105:89-96
© 2007 International Anesthesia Research Society
doi: 10.1213/01.ane.0000263030.13249.36
ANESTHETIC PHARMACOLOGY
The Role of Protein Kinase A in Acute Ethanol-Induced Neurobehavioral Actions in Rats
Chih-Chia Lai, PhD*
,
Ting-In Kuo, MS, and
Hsun-Husn Lin, PhD
From the Departments of *Pharmacology and Physiology, Institute of Pharmacology and Toxicology, Tzu Chi University, Hualien, Taiwan.
Address correspondence and reprint requests to Hsun-Husn Lin, PhD, Department of Physiology, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien, Taiwan 970. Address e-mail to hlin{at}mail.tcu.edu.tw.
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Abstract
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BACKGROUND: cAMP-dependent protein kinase (PKA) signaling pathways are involved in the regulation of ethanol-induced sedative effects in knockout mouse models. In the present study, we examined the role of PKA on the behavioral action caused by ethanol in Sprague Dawley rats.
METHODS: A loss of righting reflex (LORR) test was used to study the acute sedative effects of intraperitoneally injected ethanol. Rotarod performance was used to study the motor impairment caused by ethanol. Convulsions induced by intracerebroventricular (ICV) N-methyl-d-aspartate (NMDA) were used to evaluate ethanol’s effect on NMDA receptors. Western blot analysis was used to assay protein levels for NR1 and phosphoserine 897 on NR1 subnuits.
RESULTS: ICV pretreatment with H-9 (a nonspecific PK inhibitor) or KT 5720 (a specific PKA inhibitor) dose-dependently attenuated ethanol-induced sleeping time as assessed by LORR. ICV KT 5720 did not reduce ketamine or pentobarbital-induced sleeping time. Pretreatment with forskolin (an activator of adenylyl cyclase) or chelerythrine (a selective PKC inhibitor) had no effect on ethanol-induced LORR. Ethanol-induced motor impairment was also attenuated after pretreatment with KT 5720. Ethanol significantly inhibited NMDA-induced convulsions; the inhibitory effects of ethanol were reduced by prior ICV KT 5720, which had no significant effects on the levels of phosphoserine 897 on NMDA NR1 subunits in the several brain areas we examined.
CONCLUSIONS: Our results suggest that the PKA pathway may participate in ethanol-induced neurobehavioral changes and that NMDA receptors may be involved in the PKA regulation of ethanol’s actions.
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Introduction
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Ethanol acts as a central nervous system depressant, causing many neurobehavioral changes such as sedation and loss of motor coordination. Ethanol’s inhibition of neuronal activity is generally thought to result, at least partly, from its depression of overall synaptic activity by modulating the function of ligand-gate channels, especially N-methyl-d-aspartate (NMDA) and 
-aminobutyric acid A (GABAA) receptors (1). Ethanol is an NMDA receptor antagonist. Acute ethanol exposure at pharmacological concentrations inhibited NMDA receptor function in various neurons in vitro and in vivo (2–6). It has been suggested that NMDA receptors are involved in several ethanol-mediated behavioral changes in animal and clinical studies (7,8); some ethanol neurobehavioral actions may be due to inhibition of NMDA receptor function.
To elucidate the mechanisms underlying ethanol-induced behavioral changes, a number of knockout mouse models have been developed and tested for behavioral sensitivity to ethanol. Using these models, a cAMP-dependent protein kinase (PKA) signaling transduction pathway has been shown to play an important role in the modulation of several ethanol-induced behavioral actions such as sedation (9–12). PKA regulatory IIß subunit mutant mice, which exhibit lower total cAMP-stimulated PKA activity, were found to be less sensitive to the sedative effects of ethanol as assessed by loss of righting reflex (LORR) test (9). Adenylyl cyclase (AC), that converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP), is an essential component of the cAMP pathway. One study demonstrated that mice lacking pituitary adenylyl cyclase-activating polypeptide (PACAP), which would be expected to result in decreases of cAMP levels and PKA activity, exhibited resistant to ethanol-induced sedation (12). However, mice with heterozygous inactivation of the stimulatory G-protein subunit (GS
), and subsequently a reduction of AC activity, were found to be more sensitive to ethanol-induced sedation (10). A report also showed an increase in the sensitivity to ethanol-induced sleep in mice with genetic deletion of calcium-stimulated AC1 and AC8 (11). Thus, even though these studies suggest a connection between ethanol’s sensitivity and cAMP-PKA signaling pathways, the effects of decreases in cellular PKA activity on ethanol-induced sedation were not consistent.
Our recent in vivo study showed that pretreatment with PKA inhibitors reduced the inhibitory effects of ethanol on pressor effects induced by NMDA microinjected into the rostral ventrolateral medulla in adult Sprague Dawley (SD) rats (13), suggesting a role of PKA in regulating ethanol’s sensitivity of NMDA receptors. We hypothesized that the PKA signaling pathway may also regulate ethanol’s neurobehavioral action. We examined the effects of PK inhibitors applied intracerebroventricularly on ethanol-induced sedative, uncoordinated and hypothermic effects, as well as on ethanol inhibition of NMDA-mediated behavioral changes.
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METHODS
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Animals
A breeding colony of SD rats, purchased from National Laboratory Animal Breeding and Research Center (Taipei, Taiwan), was established at the Laboratory Animal Center, Tzu Chi University. Adult rats of either sex weighing 250–280 g were selected from the colony for use in the present study. Male rats were used in the test of LORR and western blotting analysis; female rats were used in the other tests. All animal procedures and protocols used in this study were reviewed and approved by the Institutional Animal Care and Use Committee of Tzu Chi University.
Intracerebroventricular Cannula Implantation
Under intraperitoneal (IP) injection of pentobarbital anesthesia (45 mg/kg), rats were placed prone in a David Kopf stereotaxic frame with the incisor bar, and an occipital hole was drilled to the desired position in relation to the bregma, as described below. A stainless steel guide cannula (23-gauge) was stereotaxically placed into the right lateral cerebroventricle using the following stereotaxic coordinates: 1.6 mm lateral to the midline, 0.8 mm caudal to the bregma, and 3.5 mm below the dorsal surface of the brain. The cannula was secured to the skull using two stainless steel screws and dental cement and closed with a removable stylet. Animals were allowed a 1-wk recovery period before testing. For ICV injections, a 27-gauge stainless steel micropipette was introduced into the ventricle through the cannula and connected to Hamilton microsyringes (50 µL); drugs (5 µL) were injected using a syringe pump at a rate of 10 µL/min. To determine correct placement of the guide cannula, the drinking behavior caused by ICV angiotensin II (50 ng, 5 µL) 3 days after surgery was observed (14); the rats that started drinking within 3 min after the injection were used for the following experiments. The percentage of successful ICV cannula was 85% (n = 246 rats).
Measurement of Duration of the LORR
Rats were injected IP with ethanol (3 g/kg, 40% wt/vol), ketamine (150 and 300 mg/kg), or pentobarbital (40 mg/kg). After the rats lost the righting reflex, they were put on their backs in their home cage. The duration of LORR was defined as the time from the loss of the righting reflex to the time at which it was regained. Animals were judged to have regained their righting reflex when they could right themselves three times within 1 min after being placed on their backs. To examine the effects of kinase inhibitors on anesthetic induced sleep time, ICV test drug or vehicle (5 µL) was administered 20 min before the administration of the anesthetics.
Rotarod Motor Coordination Test
Motor coordination was assessed by means of an automated rotarod apparatus (TSE systems, Bad Homburg, Germany). Before the test, the rats were trained on rotarod cylinder rotated at 10 rpm for at least 5 min. If the rats fell off during this training period, they were placed back on the cylinder. Three trials per rat were conducted on each of 3 consecutive days. The rats able to spend at least 3 min on the cylinder, rotated at 10 rpm, were used for the experiments. Rats were then injected IP with ethanol (1.5 g/kg, 40% wt/vol) and placed on the cylinder, which was then accelerated for 3 min, from 2.5 to 25 rpm. The time until the rats fell off the rotating cylinder was recorded. The rats were tested on the accelerating rotarod at 15, 30, 45, 60, and 75 min after injection of ethanol. To examine the effects of a protein kinase A inhibitor on ethanol-induced uncoordinated effects, KT 5720 or vehicle (5 µL) was administered intracerebroventricularly 20 min before the application of ethanol.
Measurement of Rectal Temperature
Rectal temperature was recorded with a digital thermometer (Singa Technology, Taipei, Taiwan). A probe was inserted into the rectum (about 2 cm) and left in place until a stable temperature recording was obtained (approximately 30–60 s) before and at 10, 20, 30, 40, 50, and 60 min after an IP injection of ethanol (1.5 g/kg, 40% wt/vol). To examine the effects of a protein kinase A inhibitor on ethanol-induced hypothermia, KT 5720 or vehicle (5 µL) was administered intracerebroventricularly 20 min before the administration of ethanol. Rectal temperature did not change significantly after an IP saline injection.
NMDA-Induced Convulsion
NMDA at a dose of 100 nmol (10 µL) was applied intracerebroventricularly. NMDA-induced convulsions were measured at 20 s after the injection and scored according to a five-point scale (0–5): 0, no convulsions; 1, slight excitation; 2, escape reaction; 3, strong escape reaction with occasional falling; 4, clonic seizures; 5, tonic seizures (15). To examine whether ethanol or pentobarbital affects NMDA-induced convulsion, the anesthetic was injected IP 30 min before the application of NMDA. To examine the effect of a protein kinase A inhibitor on the anesthetics action, ICV KT 5720 or vehicle (5 µL) was administered 20 min before the application of the anesthetics.
Western Blot Analysis
Rats were killed 20 min after ICV injection of KT 5720, calyculin A or vehicle. Brains were rapidly removed and dissected into regions of brainstem, cerebellum, hippocampus, and cerebral cortex. They were frozen in liquid nitrogen and stored at –85°C until use. About 50 mg of tissue was homogenized in 2 mL solution (0.32 M sucrose, 1 mM EDTA and 1 mTIU/mL aprotinin) with a homogenizer at speed of 10,000 rpm for 2 x 30 s. SDS was added to the sample to a final concentration of 0.1%, and 20 µg of protein was electrophoresed on 8% denaturing polyacrylamide gels. Separated proteins were transferred to nitrocellulose membrane and probed with primary antibody, anti-NR1 (1:2000, Upstate, Lake Placid, NY) or anti-phospho-NR1 (Ser897) (1:2000, Upstate). Bound antibody was incubated with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase which was reacted with Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA). The chemiluminescent signal was detected by radiograph film (Fuji Photo Film CO, Tokyo), and the bands were digitalized by scanner and analyzed with UN-SCAN-IT gel software version 6.1 for Windows (Silk Scientific Corporation, Orem, UT). Protein concentrations were determined by BCA method (Sigma CO, St. Louis, MO) using bovine albumin as standard.
Chemicals
Ethanol was purchased from Riedel-de Haen (Deisenhofen, Germany). The kinase modulators, KT 5720, H-9 dihydrochloride, chelerythrine chloride, forskolin, and calyculin A, were obtained from Tocris Cookson Ltd. (Bristol, UK). KT 5720, forskolin and calyculin A were dissolved in dimethyl sulfoxide (DMSO); the other agents were dissolved in saline. Pentobarbital was from MTC Pharmaceuticals (Ontario, Canada). NMDA, ketamine, aprotinin and other reagents used for western blot analysis were purchased from Sigma CO. Reagents for electrophoresis were obtained form Bio-Rad Laboratories (Richmond, CA).
Statistical Analysis
Data are presented as mean ± sem and were plotted and analyzed statistically with GraphPad Prism version 4.00 for Windows, GraphPad Software (San Diego, CA). The results of loss of righting reflex testing and NMDA-induced convulsion were analyzed by unpaired t-test (comparison of two groups) or one-way ANOVA followed by the Bonferroni correction (comparison of three or more groups); the results of ethanol-induced uncoordination and hypothermia were analyzed using two-way ANOVA followed by the Bonferroni post-test. A value of P <0.05 was considered statistically significant.
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RESULTS
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The Effects of PK Inhibitors on Ethanol-Induced Sedation
Intraperitoneal injection of 3 g/kg ethanol (40%) induced sleep in all rats examined; the onset and duration of LORR were 2.3 ± 0.13 min and 94.3 ± 7.5 min (n = 10 rats), respectively. ICV injection of DMSO or kinase inhibitors at tested doses, as shown below, did not cause noticeable changes in rat behavior. The onset and duration of LORR in rats pretreated with DMSO after IP ethanol (3 g/kg) were 2.9 ± 0.35 min and 104.4 ± 10 min (n = 15 rats), respectively. These were not different from the results in rats without ICV injection. Rats pretreated with KT 5720, a selective protein kinase A inhibitor, 20 min before ethanol application were more resistant to ethanol-induced sedation compared to those pretreated with DMSO. The duration of LORR in most of rats pretreated with KT 5720 were reduced after ethanol injection. Some of the rats tested did not fall asleep; the percentage of the rats staying awake after ICV pretreatment with KT 5720 at doses of 2.5, 10, and 20 nmol were 27% (n = 11 rats), 40% (n = 10 rats) and 67% (n = 6 rats), respectively. The duration of LORR in these rats was recorded as 0 min. Figure 1a shows that ICV KT 5720 significantly decreased the duration of LORR after IP injection of ethanol (3 g/kg) in a dose-dependent manner. Conversely, ICV injection of KT 5720 (2.5 and 10 nmol) 5 min after the onset of LORR did not change the duration of LORR induced by ethanol (3 g/kg); ethanol-induced sleeping time in rats post-treated with DMSO, 2.5 and 10 nmol KT 5720 were 115.1 ± 4 min (n = 10 rats), 122.5 ± 7.2 min (n = 8 rats) and 122.1 ± 23 min (n = 6 rats). Figure 1b show the duration of LORR induced by ethanol in rats pretreated with H-9 dihydrochloride, a nonspecific protein kinase inhibitor; ethanol-induced sleeping time was significantly reduced in these rats in comparison to those injected with saline. The percentage of the rats that did not sleep after ICV H-9 dihydrochloride at doses of 1 and 10 nmol were 17% (n = 6 rats) and 50% (n = 8 rats), respectively. Conversely, rats pretreated with forskolin, an activator of adenylyl cyclase, did not change their sensitivity to ethanol-induced sedation compared to those injected with DMSO; the onset and duration of LORR induced by ethanol (3 g/kg) were 2.5 ± 0.5 min and 101.6 ± 3.7 min (n = 4 rats) and 2.2 ± 0.11 min and 113.7 ± 10.5 min (n = 5 rats) in rats pretreated with 10 and 100 nmol of forskolin, respectively. In addition, ICV pretreatment with chelerythrine chloride (200 nmol), a specific protein kinase C inhibitor, did not significantly change the duration of LORR induced by ethanol; the onset and duration of LORR were 3.13 ± 0.38 min and 93.7 ± 20.1 min (n = 5 rats).
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Figure 1. The duration of loss of righting reflex induced by IP injected ethanol (3 g/kg, 40%) in rats pretreated with KT 5720, a selective cAMP-dependent protein kinase inhibitor (a), or H-9 dihydrochloride, a nonspecific protein kinase inhibitor (b). KT 5720 or H-9 dihydrochloride was applied intracerebroventricularly 20 min before the application of ethanol. The duration of LORR (sleeping time) induced by ethanol was significantly reduced in rats pretreated with KT5720 or H-9 dihydrochloride in comparison with control rats injected with DMSO or saline. Top figure (a): bars denote mean ± sem from 15, 11, 10, 6 rats treated with DMSO (control), 2.5 nmol, 10 nmol, and 20 nmol of KT 5720, respectively. Bottom figure (b): bars denote mean ± sem from 7, 6, and 8 rats treated with saline (control), 1 nmol, and 10 nmol of H-9 dihydrochloride, respectively. *Significant difference from control rats.
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Intraperitoneal injection of ketamine induced sleep in rats tested in a dose-dependent manner. The duration of LORR in rats injected with 150 and 300 mg/kg ketamine were 59.4 ± 2.4 min (n = 4 rats) and 97.2 ± 5.5 min (n = 4 rats), respectively. ICV pretreatment with KT 5720 (10 nmol) did not cause any significant change in ketamine-induced sleep time as shown in Figure 2a. In addition, pretreatment with KT 5720 did not change the duration of LORR induced by IP injection of pentobarbital (40 mg/kg) as shown in Figure 2b.
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Figure 2. Effect of KT 5720 (KT), a selective protein kinase A inhibitor, on the duration of loss of righting reflex (LORR) induced by IP injected ketamine (Ket) (a) or pentobarbital (b). KT 5720 was applied intracerebroventricularly 20 min before the application of ketamine or pentobarbital. Pretreatment with KT 5720 had no significant effects on ketamine- or pentobarbital-induced sleeping time. Top figure (a): bars denote mean ± sem from four to five rats injected with 150 mg/kg ketamine and four and six rats injected with 300 mg/kg ketamine after pretreatment with DMSO (control) and KT 5720, respectively. Bottom figure (b): bars denote mean ± sem from six and six rats treated with DMSO (control) and KT 5720, respectively.
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The Effect of KT 5720 on Ethanol-Induced Uncoordination
All rats tested were able to remain on the rotarod 180 s. Injection of small dose of ethanol (1 g/kg) did not significantly change rotarod performance over 75 min period of observation; the rats remained on the cylinder 146 ± 17.9 s, 160 ± 9.2 s, 171 ± 9.2 s, 170 ± 6.2 s, and 180 ± 0 s (n = 6 rats, P > 0.05 analyzed by repeated measure ANOVA) at 15, 30, 45, 60, and 75 min, respectively, after the injection. Higher dose of ethanol (1.5 g/kg) caused a significant impairment in rotarod performance as shown in Figure 3; the effects lasted for 60 min. ICV pretreatment with KT 5720 (2.5 and 10 nmol) significantly reduced ethanol-induced uncoordinated effects. Figure 3 show the time courses of ethanol-induced uncoordination as assessed by rotarod test in rats pretreated with DMSO and two doses of KT 5720, where KT 5720 significantly attenuated ethanol-induced impairment in rotarod performance at 30 and 45 min after injection of ethanol.
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Figure 3. The effects of KT-5720 (KT), a selective protein kinase inhibitor, on ethanol-induced motor impairment as assessed by rotarod test. Rats without treatment were able to stay on rotated cylinder for at least 180 s. Intraperitoneal injection of ethanol (1.5 g/Kg) caused a significant decrease in the time (s) that rat remained on rotarod over the observation period (75 min). Ethanol-induced uncoordinated effect was attenuated after pretreatment with KT5720 (2.5 and 10 nmol), which was applied intracerebroventricularly 20 min before the application of ethanol. Bars denote mean ± sem from 17, 7, and 14 rats treated with DMSO (control), 2.5, and 10 nmol of KT 5720, respectively. *Significant difference to pre-injection performance as analyzed with the repeated measure ANOVA followed by Bonferroni posttest. #Significant difference from control rats treated with DMSO (open bar) as analyzed with two-way ANOVA followed by Bonferroni posttests.
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The Effect of KT 5720 on Ethanol-Induced Hypothermia
Intraperitoneal injection of ethanol (1.5 g/kg) lowered body temperature within 10 min, and the effects reached the maximum by 30 min after injection. ICV pretreatment with DMSO or KT 5720 (10 nmol) 20 min before the injection of ethanol did not cause significantly changes in ethanol-induced hypothermic effects.
The Effect of KT 5720 on Ethanol Inhibition of NMDA-Induced Convulsion
ICV NMDA (100 nmol) produced convulsion in rats shortly after the injection; the intensity of convulsion reached the score of three within 20 s after the injection in all rats examined (n = 8 rats). The convulsion intensity increased to higher score (5) 10–20 min later in 50% of rats examined. Intraperitoneal injection of ethanol inhibited convulsion intensity induced by NMDA in a dose-dependent manner; the scores of convulsion were 1.1 ± 0.46 (n = 10 rats) and 0.33 ± 0.21 (n = 6 rats) after injection of 1.5 and 2.5 g/kg ethanol, respectively, whereas convulsion intensity was measured at 20 s after injection of NMDA. ICV pretreatment with KT 5720 (10 nmol) significantly reduced ethanol (1.5 g/kg) inhibition of NMDA-induced convulsion intensity, as shown in Table 1. Intraperitoneal injection of pentobarbital (30 and 40 mg/kg) dose-dependently inhibit NMDA-induced convulsion intensity; the score of NMDA-induced convulsion 30 min after injection of pentobarbital (30 mg/kg) was 0.8 ± 0.2 in rats ICV pretreated with DMSO (n = 5 rats). ICV pretreatment with KT 5720 (10 nmol) had no significant effects on pentobarbital inhibition of NMDA-induced convulsion.
The Effects of KT 5720 and Calyculin A on Phosphoserine 897 on NR1 Subunit in Brains
The protein content of NR1 subunit and the level of phosphoserine 897 on NR1 subunit in various brain areas before and 20 min after ICV KT 5720 were estimated by immunological staining of western blots with antibody against NR1 subunit and phosphorylated serine 897 on the NR1 subunit. Rats pretreated with 10 nmol of KT 5720, which reduced several ethanol neurobehavioral effects, did not show a significant change in the levels of phosphoserine 897 on NR1 subunit or in the content of NR1 protein in the areas of brainstem, cortex, cerebellum, and hippocampus in comparison to those treated with DMSO (n = 3 rats). Conversely, ICV pretreatment with 50 pmol calyculin A, a protein phosphatases inhibitor, resulted in a significant increase of phosphoserine 897 on NR1 subunit in brainstem and cerebellum, but not in the cortex and hippocampus. There is no significant difference in NR1 protein levels in all brain areas tested between control rats and calyculin-A-treated rats. In rats pretreated with calyculin A, the ratio of the concentrations of phosphoserine 897 on NR1 subunit to the protein contents of NR1 subunit increased by 59% ± 17% and 29% ± 12% (n = 3 rats) in brainstem and cerebellum, respectively, in comparison with those treated with DMSO. It is interesting that ICV application of 50 pmol calyculin A elicited an exciting behavior such as rolling (n = 4 rats). The representative western blot examining the effects of KT 5720 and calyculin A on the levels of phosphoserine 897 on NR1 subunit is shown in Figures 4a and b, respectively.
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Figure 4. Western blot analysis of the levels of phosphoserine 897 on NR1 subunit (pNR1, top) and NR1 subunits (NR1, bottom) in brainstem (BS), cortex (CTX), cerebellum (CB), and hippocampus (Hipp) of rats treated with 10 nmol KT 5720 (a) and 50 pmol calyculin A (b). The – and + represent samples from control rats injected with DMSO and rats injected with KT 5720 or calyculin A, respectively. The protein content of NR1 subunit in each sample is used as an internal control. Intracerebroventricular pretreatment with calyculin A resulted in a significant increase of phosphoserine 897 on NR1 subunit in brainstem and cerebellum, but not in the cortex and hippocampus. Rats pretreated with KT 5720 did not show a significant change in the levels of phosphoserine 897 on NR1 subunit and the content of NR1 protein in all areas tested.
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DISCUSSION
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Previously, we found that pretreatment with PKA inhibitors inhibited the ethanol inhibition of NMDA-mediated pressor effects in adult SD rats (13). The current study was designed to determine whether a decrease in brain PKA activity is also an important factor involved in the regulation of ethanol-induced neurobehavioral changes. We found that ICV pretreatment with PKA inhibitors, at doses without causing noticeable changes in rat behavior, significantly reduced acute ethanol-induced sedative and uncoordinated effects. Moreover, pretreatment with KT 5720 also attenuated ethanol inhibition of NMDA-induced convulsion. The present study shows the first data of the blockade by PKA inhibitors of several ethanol neurobehavioral effects, and provides evidence for a critical role of PKA in regulation of acute ethanol depression on CNS.
Because animals displayed hypnotic tolerance after repeated administrations of ethanol (16), each rat tested was injected with ethanol only once during this study. In addition, male rats were used in the test of LORR because they are more sensitive to the behavioral sedative effect of ethanol than female rats (17,18). ICV pretreatment with H-9, a nonspecific protein kinase inhibitor inhibiting several kinds of protein kinases such as PKA and PKC, or KT 5720, a selective PKA inhibitor, reduced ethanol-induced sleep time in a dose-dependent manner. The effects of H-9 on ethanol-induced sedation may result mainly from its inhibition of PKA as chelerythrine, a selective PKC inhibitor, had no significant effects on ethanol-induced sedation. Ketamine and pentobarbital-induced sleep was not antagonized by a PKA inhibitor, suggesting a selective regulation by PKA of ethanol-induced sedation.
Our results are complementary to the findings from several knockout mouse models in which mice with deficit in PKA regulatory IIß subunit or PACAP were more resistant to ethanol-induced sleep (9,12). Although pretreatment with KT 5720 blocked ethanol-induced sleep, it loses its inhibition while applied 5 min after the onset of ethanol-induced LORR, suggesting that PKA is only involved in the initiation of ethanol action. It is possible that PKA-regulated downstream targets participate in the regulation of ethanol sedative effects. PKA inhibitors block ethanol action by interfering with the interaction between ethanol and the targets. Once ethanol acts at the targets, PKA inhibitors lose the effects. Based on the proposed mechanism, the lack of effects of forskolin, an AC activator, on ethanol-induced sleep in our results indicates that increases in cAMP-PKA signal may not influence ethanol sensitivity to the targets.
We also examine the involvement of PKA on ethanol-induced uncoordinated effects, and showed for the first time that the uncoordinated induced by ethanol was significantly attenuated by pretreatment with KT 5720. Rats pretreated with KT 5720 regained their motor ability much sooner than control rats after IP injection of ethanol. Conversely, pretreatment with KT 5720 had no significantly effects on ethanol-induced hypothermia. It has been shown that mice defective in the AMPA receptor subunit GLuR1, which exhibited no difference in ethanol sedative or uncoordinated effects, are not sensitive to ethanol-induced hypothermia in comparison with wild-type mice. This study and ours suggest that the mechanisms underlying ethanol-induced hypothermic effects and those underlying ethanol-induced neurobehavioral effects are different. As ethanol-induced hypothermia has been suggested to be involved with both central and peripheral mechanisms (19), this may partly explain the lack of significant effects of ICV PKA inhibitors on ethanol-induced hypothermia in the present study. Similar to the results from a previous report by Danysz et al. (15), the convulsions induced by ICV NMDA are antagonized by ethanol in the present study. We also showed that pretreatment with PKA inhibitors reduced ethanol inhibitory effects on NMDA-induced convulsion. In animal study, an inhibition of NMDA receptor function by NMDA receptor antagonists effectively reduced ethanol relapse behavior (20). Clinical studies also suggest an important role of NMDA receptors in mediating some ethanol actions. Ketamine, a NMDA receptor antagonist, produced dose-related ethanol-like sedative effects in recently detoxified alcoholics (21). In addition, a recent study reported that alteration in NMDA receptor function may contribute to the risk of developing alcoholism because individuals with a family history of alcoholism were less sensitive to ketamine-induced behavioral responses (22). These studies suggest that some of ethanol behavioral actions are mediated via NMDA receptors. Therefore, the PKA regulation of ethanol effects on NMDA-induced responses may contribute to, at least partly, the inhibition of ethanol-induced neurobehavioral action by PKA inhibitors in the present study.
The phosphorylation state of serine 897 on NR1 subunit is regulated by PKA and protein phosphatases (23). In the present study, we found that the levels of phosphoserine 897 were increased by pretreatment with a phosphatase inhibitor, calyculin A, but not affected by a PKA inhibitor, KT 5720, as assessed by western blotting. This implies that the constitutive phosphatase probably dominates the phosphorylation state of the NMDA NR1 subunit under resting state in some of brain areas. KT 5720, at doses reducing ethanol neurobehavioral actions and ethanol inhibition of NMDA-induced responses, did not cause changes in the level of phosphoserine 897 on NR1 subunit, suggesting that NMDA NR1 subunit may not be the action site of PKA inhibitors. The other PKA-regulated phosphoproteins may be the targets mediating the effects of PKA inhibitors on ethanol action. cAMP-PKA signaling pathways, for example, may regulate the activation of Fyn, a nonreceptor type tyrosine kinase. Fyn has been demonstrated to mediate ethanol-induced phosphorylation of NR2B, which is involved in several forms of ethanol-induced behaviors (24–26). The precise mechanism underlying PKA regulation of ethanol actions remains to be clarified.
Many studies have suggested that the behavioral effects of ethanol are species- and age-dependent and that they may vary between female and male animals (18,27,28). The present study demonstrated that the PKA signals may be involved in the regulation of sedative effects of ethanol in male SD rats and uncoordinated effects of ethanol in female rats. Whether the PKA regulation of ethanol neurobehavioral action exerts different sensitivity in various species, ages or sexes remains unclear and deserves further study. In addition, estradiol may induce membrane-mediated PKA activation in living hippocampal neural cells (29) and cultured cerebellar granule neurons (30). Thus, there may be gender differences in neuronal PKA activity.
Rats with high alcohol consumption, which were more resistant to ethanol-induced sedation than those of less alcohol intake, have lower levels of Gs in certain brain areas (31) which is expected to result in a decrease in cAMP-PKA signaling pathway. Our results showed that rats with decreases in brain PKA activity were resistant to ethanol-induced sedation and uncoordination. Taken together, this suggests that individuals with lesser neuronal PKA activity are less sensitive to ethanol neurobehavioral action. In conclusion, our results demonstrated that decreases in brain PKA activity attenuated acute ethanol-induced sedative and uncoordinated effects and reduced the ethanol inhibition of NMDA-induced behavioral responses, suggesting that cAMP-PKA signaling pathways may play a critical role in regulating acute ethanol actions.
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ACKNOWLEDGMENTS
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We thank Mr. Kai-Tung Chuang for his assistance in ICV cannula implantation.
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Footnotes
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Accepted for publication February 26, 2007.
Supported by National Science Council grant NSC 94-2745-B-320-001 (to C.C.L.) and Tzu Chi University grant TCMRC 94013 (to H.H.L.), Taiwan.
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REFERENCES
|
|---|
- Davis KM, Wu JY. Role of glutamatergic and GABAergic systems in alcoholism. J Biomed Sci 2001;8:7–19.[ISI][Medline]
- Lin HH, Hsieh WK, Shiu JY, et al. Inhibition by ethanol of NMDA-induced responses and acute tolerance to the inhibition in rat sympathetic preganglionic neurons in vitro and in vivo. Br J Pharmacol 2003;140:955–63.[ISI][Medline]
- Lovinger DM, White G, Weight FF. Ethanol inhibits NMDA-activated ion current in hippocampal neurons. Science 1989;243:1721–4.[Abstract/Free Full Text]
- Woodward JJ. Ethanol and NMDA receptor signaling. Crit Rev Neurobiol 2000;14:69–89.[ISI][Medline]
- Lai CC, Chang MC, Lin HH. Acute tolerance to ethanol inhibition of NMDA-induced responses in rat rostral ventrolateral medulla neurons. J Biomed Sci 2004;11:482–92.[ISI][Medline]
- Li HF, Mochly-Rosen D, Kendig JJ. Protein kinase Cgamma mediates ethanol withdrawal hyper-responsiveness of NMDA receptor currents in spinal cord motor neurons. Br J Pharmacol 2005;144:301–7.[ISI][Medline]
- Kiefer F, Jahn H, Koester A, et al. Involvement of NMDA receptors in alcohol-mediated behavior: mice with reduced affinity of the NMDA R1 glycine binding site display an attenuated sensitivity to ethanol. Biol Psychiatry 2003;53:345–51.[ISI][Medline]
- Bisaga A, Evans SM. Acute effects of memantine in combination with alcohol in moderate drinkers. Psychopharmacology (Berl) 2004;172:16–24.[Medline]
- Thiele TE, Willis B, Stadler J, et al. High ethanol consumption and low sensitivity to ethanol-induced sedation in protein kinase A—mutant mice. J Neurosci 2000;20:RC75.[Abstract/Free Full Text]
- Wand G, Levine M, Zweifel L, et al. The cAMP-protein kinase A signal transduction pathway modulates ethanol consumption and sedative effects of ethanol. J Neurosci 2001;21:5297–303.[Abstract/Free Full Text]
- Maas JW Jr, Vogt SK, Chan GC, et al. Calcium-stimulated adenylyl cyclases are critical modulators of neuronal ethanol sensitivity. J Neurosci 2005;25:4118–26.[Abstract/Free Full Text]
- Tanaka K, Hashimoto H, Shintani N, et al. Reduced hypothermic and hypnotic responses to ethanol in PACAP-deficient mice. Regul Pept 2004;123:95–8.[ISI][Medline]
- Lin HH, Chang SJ, Shie HJ, Lai CC. Ethanol inhibition of NMDA-induced responses and acute tolerance to the inhibition in rat rostral ventrolateral medulla in vivo: involvement of cAMP-dependent protein kinases. Neuropharmacology 2006;51:747–55.[ISI][Medline]
- Fleegal MA, Sumners C. Drinking behavior elicited by central injection of angiotensin. II. Roles for protein kinase C and Ca2+/calmodulin-dependent protein kinase II. Am J Physiol Regul Integr Comp Physiol 2003;285:R632–40.[Abstract/Free Full Text]
- Danysz W, Dyr W, Jankowska E, et al. The involvement of NMDA receptors in acute and chronic effects of ethanol. Alcohol Clin Exp Res 1992;16:499–504.[ISI][Medline]
- Sato Y, Seo N, Kobayashi E. Ethanol-induced hypnotic tolerance is absent in N-methyl-d-aspartate receptor epsilon1 subunit knockout mice. Anesth Analg 2006;103:117–20.[Abstract/Free Full Text]
- Cha YM, Li Q, Wilson WA, Swartzwelder HS. Sedative and GABAergic effects of ethanol on male and female rats. Alcohol Clin Exp Res 2006;30:113–18.[ISI][Medline]
- Webb B, Burnett PW, Walker DW. Sex differences in ethanol-induced hypnosis and hypothermia in young Long-Evans rats. Alcohol Clin Exp Res 2002;26:695–704.[ISI][Medline]
- Kalant H, Le AD. Effects of ethanol on thermoregulation. Pharmacol Ther 1983;23:313–64.[ISI][Medline]
- Vengeliene V, Bachteler D, Danysz W, Spanagel R. The role of the NMDA receptor in alcohol relapse: a pharmacological mapping study using the alcohol deprivation effect. Neuropharmacology 2005;48:822–9.[ISI][Medline]
- Krystal JH, Petrakis IL, Webb E, et al. Dose-related ethanol-like effects of the NMDA antagonist, ketamine, in recently detoxified alcoholics. Arch Gen Psychiatry 1998;55:354–60.[Abstract/Free Full Text]
- Petrakis IL, Limoncelli D, Gueorguieva R, et al. Altered NMDA glutamate receptor antagonist response in individuals with a family vulnerability to alcoholism. Am J Psychiatry 2004;161:1776–82.[Abstract/Free Full Text]
- Westphal RS, Tavalin SJ, Lin JW, et al. Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex. Science 1999;285:93–6.[Abstract/Free Full Text]
- Yaka R, Phamluong K, Ron D. Scaffolding of Fyn kinase to the NMDA receptor determines brain region sensitivity to ethanol. J Neurosci 2003;23:3623–32.[Abstract/Free Full Text]
- Yaka R, Tang KC, Camarini R, et al. Fyn kinase and NR2B-containing NMDA receptors regulate acute ethanol sensitivity but not ethanol intake or conditioned reward. Alcohol Clin Exp Res 2003;27:1736–42.[ISI][Medline]
- Malinowska B, Napiorkowska-Pawlak D, Pawlak R, et al. Ifenprodil influences changes in mouse behaviour related to acute and chronic ethanol administration. Eur J Pharmacol 1999;377:13–19.[ISI][Medline]
- Ott JF, Hunter BE, Walker DW. The effect of age on ethanol metabolism and on the hypothermic and hypnotic responses to ethanol in the Fischer 344 rat. Alcohol Clin Exp Res 1985;9:59–65.[ISI][Medline]
- Cunningham CL, Niehus JS, Noble D. Species difference in sensitivity to ethanol’s hedonic effects. Alcohol 1993;10:97–102.[ISI][Medline]
- Shingo AS, Kito S. Estradiol induces PKA activation through the putative membrane receptor in the living hippocampal neuron. J Neural Transm 2005;112:1469–73.[ISI][Medline]
- Belcher SM, Le HH, Spurling L, Wong JK. Rapid estrogenic regulation of extracellular signal-regulated kinase 1/2 signaling in cerebellar granule cells involves a G protein- and protein kinase A-dependent mechanism and intracellular activation of protein phosphatase 2A. Endocrinology 2005;146:5397–406.[Abstract/Free Full Text]
- Froehlich JC, Wand GS. Adenylyl cyclase signal transduction and alcohol-induced sedation. Pharmacol Biochem Behav 1997;58:1021–30.[ISI][Medline]
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Abstract
Introduction
