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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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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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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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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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-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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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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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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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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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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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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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.
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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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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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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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Footnotes |
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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 |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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  M. K. Kelm, H. E. Criswell, and G. R. Breese The Role of Protein Kinase A in the Ethanol-Induced Increase in Spontaneous GABA Release Onto Cerebellar Purkinje Neurons J Neurophysiol, December 1, 2008; 100(6): 3417 - 3428. [Abstract] [Full Text] [PDF]
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