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Pretreatment with the protein kinase C activator phorbol 12,13-dibutyrate attenuates the ethanol-induced loss of the righting reflex in mice: modification by diabetes
Brain Research 764 Ž1997. 244–248
Short communication
Pretreatment with the protein kinase C activator phorbol 12,13-dibutyrate attenuates the ethanol-induced loss of the righting reflex in mice: modification by diabetes Masahiro Ohsawa, Junzo Kamei
)
Department of Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi UniÕersity, 4-41 Ebara 2-chome, Shinagawa-ku, Tokyo 142, Japan Accepted 30 April 1997
Abstract The effects of the protein kinase C ŽPKC. activator phorbol 12,13-dibutyrate ŽPDBu. on the ethanol-induced loss of the righting reflex were studied in diabetic and non-diabetic mice. The ethanol-induced loss of the righting reflex was significantly less in diabetic mice than in non-diabetic mice. Intracerebroventricular Ži.c.v.. pretreatment with PDBu dose- and time-dependently reduced the ethanol-induced loss of the righting reflex in non-diabetic mice. The reduction of the ethanol-induced loss of the righting reflex caused by PDBu was reversed by concomitant i.c.v. pretreatment with calphostin C, a selective PKC inhibitor. On the other hand, PDBu had no effect on the ethanol-induced loss of the righting reflex in diabetic mice. I.c.v. pretreatment with calphostin C Ž10 pmol. increased the ethanol-induced loss of the righting reflex in diabetic mice but not in non-diabetic mice. These results suggest that the activation of PKC reduces the ethanol-induced loss of the righting reflex in mice. Furthermore, it is possible that this attenuation of the ethanol-induced loss of the righting reflex in diabetic mice may be due in part to increased PKC activity. q 1997 Elsevier Science B.V. Keywords: Ethanol; Protein kinase C; Righting reflex; Calphostin C; Phorbol 12,13-dibutyrate; Diabetes
The behavioral effects of alcohol have been recognized for a long time and were previously attributed to an increase in the fluidity of neuronal membrane lipids. However, recent studies have suggested that the effects of ethanol are mediated by a more direct action on several different receptor proteins and ion channels; GABA, NMDA, voltage-sensitive calcium channels and 5-HT3 receptors are all influenced by intoxicating levels of alcohol w15x. The administration of ethanol to animals has also been shown to markedly decrease the turnover of phosphoinositol in the central nervous system w3,5,26x, the activity of adenylate cyclase w10x, and the levels of cAMP and cGMP in specific brain regions. A significant decrease in the baseline activity of brain PKC, a pivotal enzyme in the inositol phosphate-mediated signal transduction system, has been demonstrated in strains of mice selectively bred for sensitivity to ethanol w9x. It has also been shown that
) Corresponding author. Fax: q81 Ž3. 5498-5029; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 6 0 1 - X
therapeutic concentrations of ethanol w13x, as well as volatile general anesthetics and other central depressants Žchlorpromazine w34x and phenobarbital w6x. can substantially inhibit the activity of PKC purified from whole brain homogenates. Recently, these findings have been confirmed and extended to a wide variety of central nervous system depressants, including ethanol and other n-alkanols, in a lipid-free PKC preparation w38x. Despite these intriguing studies, the relationship between in vitro findings with PKC and other protein kinases and the in vivo effects of ethanol has not been well established. Many studies have suggested that diabetes or hyperglycemia affects the sensitivity of laboratory animals to various pharmacological agents. Several reports have demonstrated that hyperglycemic or diabetic animals show increased sensitivity to barbiturates w1,2,25,39,40x and decreased sensitivity to D-amphetamine w11,29–32x, p-chloroamphetamine w27x, carbon tetrachloride w17,18x and morphine w21,37x. Furthermore, many investigators have reported that hyperglycemia or elevated glucose levels can increase diacylglycerol ŽDAG. levels and activate protein kinase C ŽPKC. in vascular tissues, cardiac tissues, or
M. Ohsawa, J. Kameir Brain Research 764 (1997) 244–248
cultured cells w8,20,23,42x. Activation of the DAG-PKC cellular signal pathway is linked to dysfunction of the vasculature in diabetes w8,36,43x. Thus, the first aim of our study was to investigate the effect of the PKC activator phorbol 12,13-dibutyrate ŽPDBu. on the ethanol-induced loss of the righting reflex in mice. We also examined the effect of diabetes on the ethanol-induced loss of the righting reflex in mice and the possible involvement of protein kinase C activation. Male ICR mice ŽTokyo Laboratory Animals Science Co., Tokyo, Japan., weighing about 20 g at the beginning of the experiments, were used. They had free access to food and water in an animal room which was maintained at 24 " 18C with a 12 h light–dark cycle. Animals were rendered diabetic by an injection of streptozotocin ŽSTZ; 200 mgrkg, i.v.. prepared in 0.1 N citrate buffer at pH 4.5. Age-matched non-diabetic mice were injected with vehicle alone. The experiments were conducted 2 weeks after the injection of STZ or vehicle. Mice with serum glucose levels above 400 mgrdl were considered diabetic. This study was carried out in accordance with the Declaration of Helsinki andror with the Guide for the Care and Use of Laboratory Animals as adopted by the Committee on the Care and Use of Laboratory Animals of Hoshi University, which is accredited by the Ministry of Education, Science, Sports and Culture of Japan. The duration of the loss of the righting reflex response was measured according to the procedures described by Marley et al. w28x. When the animals become ataxic after i.p. injection of ethanol, they were placed in a V-shaped plastic trough and the time was recorded. The animals were considered to have regained the righting response after they righted themselves 3 times in 30 s. The amount of time between the loss and regaining of the response was rounded to the nearest minute. To avoid problems associated with toxicity, we attempted to find doses that elicited sleep times of 180 min or less w28x. Streptozotocin ŽSTZ. was purchased from Sigma Chemical Co., St. Louis, MO, USA. Phorbol 12,13-dibutyrate ŽPDBu. was purchased from Research Biochemicals, Natick, MA, USA. Calphostin C was purchased from Calbiochem-Novabiochem International, San Diego, CA, USA. STZ was dissolved in 0.1 N citrate buffer. PDBu and calphostin C were dissolved in ethanol 0.01% in saline. PDBu and calphostin C were injected intracerebroventricularly Ži.c.v... I.c.v. administration Ž5 m lrmouse. was performed according to the method described by Haley and McCormic w16x using a 50 m l Hamilton syringe. Although responses to ethanol have been shown to depend on the injection concentration w14x, the effect was only observed at concentrations above 20% vrv. Data are expressed as the mean with S.E. The statistical significance of differences was assessed by a one-way analysis of variance ŽANOVA. with repeated measures followed by Dunnett’s test. A level of probability of 0.05 or less was considered significant.
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Fig. 1. A: time course of the effect of i.c.v. pretreatment with phorbol 12,13-dibutyrate ŽPDBu; 50 pmol, hatched column. on the duration of the ethanol ŽEtOH; 4 grkg, i.p..-induced loss of the righting reflex in non-diabetic mice. B: dose-response effects of PDBu on the duration of the EtOH-induced loss of the righting reflex in non-diabetic mice. C: effect of calphostin C Ždotted column. on the PDBu Ž50 pmol.-induced reduction in the duration of the EtOH Ž4 grkg, i.p..-induced loss of the righting reflex in non-diabetic mice. PDBu and vehicle were injected i.c.v. 0.5, 1, 2, 4 h ŽA. or 1 h ŽB and C. before the administration of EtOH. Calphostin C or its vehicle Žcross-hatched column. was co-administered with PDBu 1 h prior to i.p. administration of EtOH. Each column represents the mean with S.E. of 10–12 animals. ) P - 0.05 compared to the vehicle in PDBu pretreatment Žopen column.. a P - 0.05 compared to the vehicle in calphostin C pretreatment Žcross-hatched column..
In non-diabetic mice, pretreatment with PDBu Ž50 pmol, i.c.v.. for 60, 120 and 240 min, but not for 30 min, reduced the duration of the ethanol Ž4 grkg.-induced loss of the righting reflex ŽFig. 1A.. The effects of various doses of PDBu on the ethanol-induced loss of the righting reflex in non-diabetic mice are shown in Fig. 1B. Pretreatment with PDBu for 60 min, at doses of 10–50 pmol, reduced the duration of the ethanol-induced loss of the righting reflex in non-diabetic mice in a dose-dependent manner. The reduction of the duration of the ethanol Ž4 grkg.-induced loss of the righting reflex by PDBu Ž50 pmol. was dose-dependently reversed by concomitant pretreatment with calphostin C, at doses of 1–10 pmol ŽFig. 1C..
246
M. Ohsawa, J. Kameir Brain Research 764 (1997) 244–248
Fig. 2. A: duration of the ethanol ŽEtOH.-induced loss of the righting reflex in diabetic Žhatched column. and non-diabetic mice Žopen column.. B: effect of phorbol 12,13-dibutyrate ŽPDBu; 50 pmol, hatched column. on the duration of the EtOH Ž4 and 4.4 grkg, i.p..-induced loss of the righting reflex in diabetic mice. C: effect of calphostin C ŽCP; 10 pmol, hatched column. on the duration of the EtOH Ž4 grkg, i.p..-induced loss of the righting reflex in diabetic mice and non-diabetic mice. PDBu, calphostin C or vehicle were injected i.c.v. 60 min before the administration of ethanol. Each column represents the mean with S.E. of 8–12 animals. ) P - 0.05 compared to non-diabetic mice. a P - 0.05 compared to pretreatment with the vehicle.
As shown in Fig. 2A, intraperitoneal injection of ethanol Ž3.2 and 4.0 grkg. produced a dose-dependent increase in the duration of the loss of the righting reflex in both diabetic and non-diabetic mice. Diabetic mice were significantly less sensitive to ethanol than non-diabetic mice. Furthermore, in diabetic mice, PDBu had no significant effect on the duration of the ethanol-induced loss of the righting reflex Ž4.0 and 4.4 grkg; Fig. 2B.. As shown in Fig. 2C, pretreatment with calphostin C, at a dose of 10 pmol, i.c.v., for 60 min significantly increased the duration of the loss of the righting reflex produced by ethanol Ž4 grkg. in diabetic mice. However, calphostin C, by itself, had no effect on the duration of the ethanol Ž4 grkg.-induced loss of the righting reflex in non-diabetic mice. In the present study, we demonstrated that i.c.v. pretreatment with PDBu Ž50 pmol. attenuated the duration of the ethanol-induced loss of the righting reflex in non-di-
abetic mice. Furthermore, this attenuation of the duration of the ethanol-induced loss of the righting reflex by PDBu was reversed by concomitant i.c.v. pretreatment with calphostin C, a selective PKC inhibitor. These results provide evidence that the attenuation of the ethanol-induced loss of the righting reflex by PDBu is specifically mediated by the activation of PKC and suggest that central PKC activation may attenuate the ethanol-induced loss of the righting reflex. This possibility is supported by the suggestion that central protein kinase inhibition may be involved in the acute intoxicating effects of ethanol, since staurosporine, a potent PKC inhibitor, increases the ethanol-induced loss of the righting reflex in Rana pipiens tadpoles w12x. On the other hand, Harris et al. w19x reported that mutant mice which lacked the g isoform of PKC displayed reduced sensitivity to the effect of ethanol on the righting reflex. Thus far, seven isoforms of PKC, i.e., a , b I, b II, g , d , e and j , have been identified. Furthermore, it has been reported that the g and a isoforms are activated much less by diacylglycerol in the presence of phosphatidylserine than a mixture of the b I and b II isoforms w35x. In the present study, the attenuation of the duration of the ethnol-induced loss of the righting reflex by PDBu was reversed by concomitant pretreatment with calphostin C, which specifically inhibits the binding of DAG to the regulatory domain of PKC w24x. Moreover, it has been reported that intravitreal injection of PDBu produces a significant increase in b II isoforms, but not other isoforms, in the membranous pool in retinas w36x. Thus, the attenuation of the duration of the ethanol-induced loss of the righting reflex caused by PDBu may be due to the activation of the b I andror b II isoforms of PKC. However, further studies are necessary before this possibility can be established with greater certainty. The mechanisms which underlie the attenuation of the duration of the ethanol-induced loss of the righting reflex by PDBu are unclear. It has been reported that the effects of ethanol are mediated by more its direct action on several different receptor proteins and ion channels; GABA, NMDA, voltage-sensitive calcium channels and 5-HT3 receptors are all influenced by intoxicating levels of alcohol w15x. There is accumulating evidence that the activation of PKC regulates several cellular functions through the phosphorylation of proteins, including some receptors, whose function is then down-regulated w4,22,33,41x. Our present results suggest that the action sites of ethanol may be desensitized by the phosphorylation induced by activated PKC. Furthermore, it is important to observe that at least 60 min of pretreatment was required for the PDBu-induced attenuation of the ethanol-induced loss of the righting reflex. Pretreatment with PDBu for 30 min or less did not attenuate the duration of the ethanol-induced loss of the righting reflex. On the other hand, it has been reported that ethanol affects intracellular messengers w3,5,10,13,26,38x. It has been reported that ethanol inhibits PKC activity and treatment with 12-O-tetradecanoylphorbol-13-acetate an-
M. Ohsawa, J. Kameir Brain Research 764 (1997) 244–248
tagonizes the ethanol-induced inhibition of PKC activity w38x. Furthermore, activation of PKC by phorbol 12-myristate 13-acetate inhibits the ethanol-induced inhibition of adenosine uptake in NG 108-15 cells w7x. These results support the possibility that the pharmacological actions of ethanol are inhibited by the activation of PKC by phorbol ester. In the present study, we also demonstrated that the duration of the ethanol-induced loss of the righting reflex in diabetic mice was significantly shorter than that in non-diabetic mice. Moreover, PDBu had no significant effect on the ethanol-induced loss of the righting reflex in diabetic mice. Pretreatment with calphostin C Ž10 pmol, i.c.v.., by itself, had no significant effect on the duration of the ethanol-induced loss of the righting reflex in non-diabetic mice. However, calphostin C significantly increased the duration of the ethanol-induced loss of the righting reflex in diabetic mice. Calphostin C specifically inhibits the binding of DAG to the regulatory domain of PKC w24x and therefore is a more selective inhibitor than staurosporine or H-7, which interact with the ATP-binding site of PKC that shares substantial homology with other protein kinases. These results suggest that the attenuation of the duration of the ethanol-induced loss of the righting reflex in diabetic mice may be, in part, due to the increased activity of PKC. In conclusion, these results suggest that central PKC may be involved in the ethanol-induced loss of the righting reflex. Furthermore, the different sensitivity to ethanol in diabetic mice may be partly due to the enhanced activity of PKC.
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Acknowledgements We wish to thank Ms. M. Hanamura and M. Itoh for their helpful technical assistance.
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References
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Short communication
Pretreatment with the protein kinase C activator phorbol 12,13-dibutyrate attenuates the ethanol-induced loss of the righting reflex in mice: modification by diabetes Masahiro Ohsawa, Junzo Kamei
)
Department of Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi UniÕersity, 4-41 Ebara 2-chome, Shinagawa-ku, Tokyo 142, Japan Accepted 30 April 1997
Abstract The effects of the protein kinase C ŽPKC. activator phorbol 12,13-dibutyrate ŽPDBu. on the ethanol-induced loss of the righting reflex were studied in diabetic and non-diabetic mice. The ethanol-induced loss of the righting reflex was significantly less in diabetic mice than in non-diabetic mice. Intracerebroventricular Ži.c.v.. pretreatment with PDBu dose- and time-dependently reduced the ethanol-induced loss of the righting reflex in non-diabetic mice. The reduction of the ethanol-induced loss of the righting reflex caused by PDBu was reversed by concomitant i.c.v. pretreatment with calphostin C, a selective PKC inhibitor. On the other hand, PDBu had no effect on the ethanol-induced loss of the righting reflex in diabetic mice. I.c.v. pretreatment with calphostin C Ž10 pmol. increased the ethanol-induced loss of the righting reflex in diabetic mice but not in non-diabetic mice. These results suggest that the activation of PKC reduces the ethanol-induced loss of the righting reflex in mice. Furthermore, it is possible that this attenuation of the ethanol-induced loss of the righting reflex in diabetic mice may be due in part to increased PKC activity. q 1997 Elsevier Science B.V. Keywords: Ethanol; Protein kinase C; Righting reflex; Calphostin C; Phorbol 12,13-dibutyrate; Diabetes
The behavioral effects of alcohol have been recognized for a long time and were previously attributed to an increase in the fluidity of neuronal membrane lipids. However, recent studies have suggested that the effects of ethanol are mediated by a more direct action on several different receptor proteins and ion channels; GABA, NMDA, voltage-sensitive calcium channels and 5-HT3 receptors are all influenced by intoxicating levels of alcohol w15x. The administration of ethanol to animals has also been shown to markedly decrease the turnover of phosphoinositol in the central nervous system w3,5,26x, the activity of adenylate cyclase w10x, and the levels of cAMP and cGMP in specific brain regions. A significant decrease in the baseline activity of brain PKC, a pivotal enzyme in the inositol phosphate-mediated signal transduction system, has been demonstrated in strains of mice selectively bred for sensitivity to ethanol w9x. It has also been shown that
) Corresponding author. Fax: q81 Ž3. 5498-5029; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 6 0 1 - X
therapeutic concentrations of ethanol w13x, as well as volatile general anesthetics and other central depressants Žchlorpromazine w34x and phenobarbital w6x. can substantially inhibit the activity of PKC purified from whole brain homogenates. Recently, these findings have been confirmed and extended to a wide variety of central nervous system depressants, including ethanol and other n-alkanols, in a lipid-free PKC preparation w38x. Despite these intriguing studies, the relationship between in vitro findings with PKC and other protein kinases and the in vivo effects of ethanol has not been well established. Many studies have suggested that diabetes or hyperglycemia affects the sensitivity of laboratory animals to various pharmacological agents. Several reports have demonstrated that hyperglycemic or diabetic animals show increased sensitivity to barbiturates w1,2,25,39,40x and decreased sensitivity to D-amphetamine w11,29–32x, p-chloroamphetamine w27x, carbon tetrachloride w17,18x and morphine w21,37x. Furthermore, many investigators have reported that hyperglycemia or elevated glucose levels can increase diacylglycerol ŽDAG. levels and activate protein kinase C ŽPKC. in vascular tissues, cardiac tissues, or
M. Ohsawa, J. Kameir Brain Research 764 (1997) 244–248
cultured cells w8,20,23,42x. Activation of the DAG-PKC cellular signal pathway is linked to dysfunction of the vasculature in diabetes w8,36,43x. Thus, the first aim of our study was to investigate the effect of the PKC activator phorbol 12,13-dibutyrate ŽPDBu. on the ethanol-induced loss of the righting reflex in mice. We also examined the effect of diabetes on the ethanol-induced loss of the righting reflex in mice and the possible involvement of protein kinase C activation. Male ICR mice ŽTokyo Laboratory Animals Science Co., Tokyo, Japan., weighing about 20 g at the beginning of the experiments, were used. They had free access to food and water in an animal room which was maintained at 24 " 18C with a 12 h light–dark cycle. Animals were rendered diabetic by an injection of streptozotocin ŽSTZ; 200 mgrkg, i.v.. prepared in 0.1 N citrate buffer at pH 4.5. Age-matched non-diabetic mice were injected with vehicle alone. The experiments were conducted 2 weeks after the injection of STZ or vehicle. Mice with serum glucose levels above 400 mgrdl were considered diabetic. This study was carried out in accordance with the Declaration of Helsinki andror with the Guide for the Care and Use of Laboratory Animals as adopted by the Committee on the Care and Use of Laboratory Animals of Hoshi University, which is accredited by the Ministry of Education, Science, Sports and Culture of Japan. The duration of the loss of the righting reflex response was measured according to the procedures described by Marley et al. w28x. When the animals become ataxic after i.p. injection of ethanol, they were placed in a V-shaped plastic trough and the time was recorded. The animals were considered to have regained the righting response after they righted themselves 3 times in 30 s. The amount of time between the loss and regaining of the response was rounded to the nearest minute. To avoid problems associated with toxicity, we attempted to find doses that elicited sleep times of 180 min or less w28x. Streptozotocin ŽSTZ. was purchased from Sigma Chemical Co., St. Louis, MO, USA. Phorbol 12,13-dibutyrate ŽPDBu. was purchased from Research Biochemicals, Natick, MA, USA. Calphostin C was purchased from Calbiochem-Novabiochem International, San Diego, CA, USA. STZ was dissolved in 0.1 N citrate buffer. PDBu and calphostin C were dissolved in ethanol 0.01% in saline. PDBu and calphostin C were injected intracerebroventricularly Ži.c.v... I.c.v. administration Ž5 m lrmouse. was performed according to the method described by Haley and McCormic w16x using a 50 m l Hamilton syringe. Although responses to ethanol have been shown to depend on the injection concentration w14x, the effect was only observed at concentrations above 20% vrv. Data are expressed as the mean with S.E. The statistical significance of differences was assessed by a one-way analysis of variance ŽANOVA. with repeated measures followed by Dunnett’s test. A level of probability of 0.05 or less was considered significant.
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Fig. 1. A: time course of the effect of i.c.v. pretreatment with phorbol 12,13-dibutyrate ŽPDBu; 50 pmol, hatched column. on the duration of the ethanol ŽEtOH; 4 grkg, i.p..-induced loss of the righting reflex in non-diabetic mice. B: dose-response effects of PDBu on the duration of the EtOH-induced loss of the righting reflex in non-diabetic mice. C: effect of calphostin C Ždotted column. on the PDBu Ž50 pmol.-induced reduction in the duration of the EtOH Ž4 grkg, i.p..-induced loss of the righting reflex in non-diabetic mice. PDBu and vehicle were injected i.c.v. 0.5, 1, 2, 4 h ŽA. or 1 h ŽB and C. before the administration of EtOH. Calphostin C or its vehicle Žcross-hatched column. was co-administered with PDBu 1 h prior to i.p. administration of EtOH. Each column represents the mean with S.E. of 10–12 animals. ) P - 0.05 compared to the vehicle in PDBu pretreatment Žopen column.. a P - 0.05 compared to the vehicle in calphostin C pretreatment Žcross-hatched column..
In non-diabetic mice, pretreatment with PDBu Ž50 pmol, i.c.v.. for 60, 120 and 240 min, but not for 30 min, reduced the duration of the ethanol Ž4 grkg.-induced loss of the righting reflex ŽFig. 1A.. The effects of various doses of PDBu on the ethanol-induced loss of the righting reflex in non-diabetic mice are shown in Fig. 1B. Pretreatment with PDBu for 60 min, at doses of 10–50 pmol, reduced the duration of the ethanol-induced loss of the righting reflex in non-diabetic mice in a dose-dependent manner. The reduction of the duration of the ethanol Ž4 grkg.-induced loss of the righting reflex by PDBu Ž50 pmol. was dose-dependently reversed by concomitant pretreatment with calphostin C, at doses of 1–10 pmol ŽFig. 1C..
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Fig. 2. A: duration of the ethanol ŽEtOH.-induced loss of the righting reflex in diabetic Žhatched column. and non-diabetic mice Žopen column.. B: effect of phorbol 12,13-dibutyrate ŽPDBu; 50 pmol, hatched column. on the duration of the EtOH Ž4 and 4.4 grkg, i.p..-induced loss of the righting reflex in diabetic mice. C: effect of calphostin C ŽCP; 10 pmol, hatched column. on the duration of the EtOH Ž4 grkg, i.p..-induced loss of the righting reflex in diabetic mice and non-diabetic mice. PDBu, calphostin C or vehicle were injected i.c.v. 60 min before the administration of ethanol. Each column represents the mean with S.E. of 8–12 animals. ) P - 0.05 compared to non-diabetic mice. a P - 0.05 compared to pretreatment with the vehicle.
As shown in Fig. 2A, intraperitoneal injection of ethanol Ž3.2 and 4.0 grkg. produced a dose-dependent increase in the duration of the loss of the righting reflex in both diabetic and non-diabetic mice. Diabetic mice were significantly less sensitive to ethanol than non-diabetic mice. Furthermore, in diabetic mice, PDBu had no significant effect on the duration of the ethanol-induced loss of the righting reflex Ž4.0 and 4.4 grkg; Fig. 2B.. As shown in Fig. 2C, pretreatment with calphostin C, at a dose of 10 pmol, i.c.v., for 60 min significantly increased the duration of the loss of the righting reflex produced by ethanol Ž4 grkg. in diabetic mice. However, calphostin C, by itself, had no effect on the duration of the ethanol Ž4 grkg.-induced loss of the righting reflex in non-diabetic mice. In the present study, we demonstrated that i.c.v. pretreatment with PDBu Ž50 pmol. attenuated the duration of the ethanol-induced loss of the righting reflex in non-di-
abetic mice. Furthermore, this attenuation of the duration of the ethanol-induced loss of the righting reflex by PDBu was reversed by concomitant i.c.v. pretreatment with calphostin C, a selective PKC inhibitor. These results provide evidence that the attenuation of the ethanol-induced loss of the righting reflex by PDBu is specifically mediated by the activation of PKC and suggest that central PKC activation may attenuate the ethanol-induced loss of the righting reflex. This possibility is supported by the suggestion that central protein kinase inhibition may be involved in the acute intoxicating effects of ethanol, since staurosporine, a potent PKC inhibitor, increases the ethanol-induced loss of the righting reflex in Rana pipiens tadpoles w12x. On the other hand, Harris et al. w19x reported that mutant mice which lacked the g isoform of PKC displayed reduced sensitivity to the effect of ethanol on the righting reflex. Thus far, seven isoforms of PKC, i.e., a , b I, b II, g , d , e and j , have been identified. Furthermore, it has been reported that the g and a isoforms are activated much less by diacylglycerol in the presence of phosphatidylserine than a mixture of the b I and b II isoforms w35x. In the present study, the attenuation of the duration of the ethnol-induced loss of the righting reflex by PDBu was reversed by concomitant pretreatment with calphostin C, which specifically inhibits the binding of DAG to the regulatory domain of PKC w24x. Moreover, it has been reported that intravitreal injection of PDBu produces a significant increase in b II isoforms, but not other isoforms, in the membranous pool in retinas w36x. Thus, the attenuation of the duration of the ethanol-induced loss of the righting reflex caused by PDBu may be due to the activation of the b I andror b II isoforms of PKC. However, further studies are necessary before this possibility can be established with greater certainty. The mechanisms which underlie the attenuation of the duration of the ethanol-induced loss of the righting reflex by PDBu are unclear. It has been reported that the effects of ethanol are mediated by more its direct action on several different receptor proteins and ion channels; GABA, NMDA, voltage-sensitive calcium channels and 5-HT3 receptors are all influenced by intoxicating levels of alcohol w15x. There is accumulating evidence that the activation of PKC regulates several cellular functions through the phosphorylation of proteins, including some receptors, whose function is then down-regulated w4,22,33,41x. Our present results suggest that the action sites of ethanol may be desensitized by the phosphorylation induced by activated PKC. Furthermore, it is important to observe that at least 60 min of pretreatment was required for the PDBu-induced attenuation of the ethanol-induced loss of the righting reflex. Pretreatment with PDBu for 30 min or less did not attenuate the duration of the ethanol-induced loss of the righting reflex. On the other hand, it has been reported that ethanol affects intracellular messengers w3,5,10,13,26,38x. It has been reported that ethanol inhibits PKC activity and treatment with 12-O-tetradecanoylphorbol-13-acetate an-
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tagonizes the ethanol-induced inhibition of PKC activity w38x. Furthermore, activation of PKC by phorbol 12-myristate 13-acetate inhibits the ethanol-induced inhibition of adenosine uptake in NG 108-15 cells w7x. These results support the possibility that the pharmacological actions of ethanol are inhibited by the activation of PKC by phorbol ester. In the present study, we also demonstrated that the duration of the ethanol-induced loss of the righting reflex in diabetic mice was significantly shorter than that in non-diabetic mice. Moreover, PDBu had no significant effect on the ethanol-induced loss of the righting reflex in diabetic mice. Pretreatment with calphostin C Ž10 pmol, i.c.v.., by itself, had no significant effect on the duration of the ethanol-induced loss of the righting reflex in non-diabetic mice. However, calphostin C significantly increased the duration of the ethanol-induced loss of the righting reflex in diabetic mice. Calphostin C specifically inhibits the binding of DAG to the regulatory domain of PKC w24x and therefore is a more selective inhibitor than staurosporine or H-7, which interact with the ATP-binding site of PKC that shares substantial homology with other protein kinases. These results suggest that the attenuation of the duration of the ethanol-induced loss of the righting reflex in diabetic mice may be, in part, due to the increased activity of PKC. In conclusion, these results suggest that central PKC may be involved in the ethanol-induced loss of the righting reflex. Furthermore, the different sensitivity to ethanol in diabetic mice may be partly due to the enhanced activity of PKC.
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Acknowledgements We wish to thank Ms. M. Hanamura and M. Itoh for their helpful technical assistance.
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References
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