- Email: [email protected]
Effect of ethanol on phosphorylation of the NMDAR2B subunit in mouse cortical neurons
Molecular Brain Research 68 Ž1999. 159–168
Research report
Effect of ethanol on phosphorylation of the NMDAR2B subunit in mouse cortical neurons Haviryaji S.G. Kalluri, Maharaj K. Ticku
)
Department of Pharmacology, The UniÕersity of Texas Health Science Center, 7703 Floyd Curl DriÕe, San Antonio, TX 78284-7764, USA Accepted 2 February 1999
Abstract There is evidence that phosphorylation plays a crucial role in the regulation of the NMDA receptors in the brain. In this study, we examined the effect of acute and chronic ethanol treatment on the phosphorylation of the R2B subunit of the NMDA receptors in fetal cortical neurons. Additionally, the effect of acute ethanol treatment on the phosphorylation of the R2B subunit in adult cerebral cortex and hippocampus was also examined. The results show that acute or chronic ethanol treatments did not affect the total phosphorylation of the R2B subunit in cortical neurons. In adult mice, we observed that acute ethanol treatment increased the tyrosine phosphorylation of the R2B subunit in hippocampus but not in cerebral cortex. We also observed that acute or chronic ethanol treatments did not alter the Fyn or Csk levels in cortical neurons. Although Fyn, but not Csk, was present in adult cerebral cortex, ethanol did not phosphorylate the R2B subunit in this region. Like ethanol, MK-801 ŽNMDA antagonist. did not affect the phosphorylation of the R2B subunit in cortical neurons. Taken together, these results suggest that acute and chronic ethanol and MK-801 treatments do not affect the phosphorylation of the R2B subunit in fetal cortical neurons and adult cerebral cortex. Based on these observations, we speculate that the R2B subunit of the NMDA receptors is regulated by multiple cascades and in a brain region specific manner. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Cultured neuron; NMDA receptor regulation; Phosphorylation; Ethanol; Fyn kinase; Csk kinase
1. Introduction The glutamate receptors mediate fast excitatory transmission in the CNS and play an important role in synaptic plasticity, neuronal development and neurological disorders w3,7,19,23x. Reversible protein phosphorylation by a number of kinases and phosphatases is a major post-translational modification, which regulates a variety of receptor proteins and their function including glutamate receptors w22,29x. Phorbol esters have been shown to increase w1x or decrease w15x NMDA receptor function. Biochemical studies have demonstrated that the NMDA receptors are phosphorylated by PKC and protein tyrosine kinases w13,25x. Earlier studies have shown that long-term potentiation ŽLTP. was blocked by tyrosine kinase inhibitors w20x, and ) Corresponding author. Fax: q1-210-567-4226; E-mail: [email protected]
that LTP-like features were elicited by injecting PKC into hippocampal neurons w8,15x. Since ethanol affects the NMDA receptor function following its acute as well as chronic administration w4– 6,9,14,27x, and that the NMDA receptor function is affected by phosphorylation, it is of interest to study whether ethanol affects the phosphorylation of the NMDA receptors. A previous study has implicated an involvement of PKC in the ethanol-induced inhibition of the NMDA responses w24x. In another study, it was shown that chronic ethanol treatment up-regulates the PKC isozymes Ž d and ´ . and PKC-mediated phosphorylation in neuronal cultures w16x. A recent study has revealed that tyrosine phosphorylation of the NMDAR2B receptor subunit was up-regulated following ethanol treatment in the hippocampus of C57BL mice and this was shown to be mediated by Fyn kinase w17x. These investigators reported that acute tolerance to ethanol in hippocampus involves tyrosine phospho-
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 0 5 7 - 1
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H.S.G. Kalluri, M.K. Tickur Molecular Brain Research 68 (1999) 159–168
rylation of the NMDA receptors by Fyn kinase w17x. Several laboratories have observed a selective and differential up-regulation of the NMDA receptor binding, function, gene, and protein expression in vivo and in vitro w2,4–6,9,27x. Additionally, chronic NMDA receptor antagonists like DŽy.-2-amino-phosphonopentanoic acid ŽAP-5. and MK-801, have been shown to produce up-regulation of NMDA receptor subunit gene and protein expression, an effect similar to that observed following chronic ethanol treatment w6x. In the present study, we investigated the effect of ethanol and MK-801 on the phosphorylation of the NMDAR2B subunit in cultured mouse cortical neurons.
drugs were replaced every 24 h. For acute studies, after 7 days in culture, neurons were treated with 75 or 150 mM for 10 or 20 min, as described below. 2.3. In ÕiÕo labeling and immunoprecipitation Prior to in vivo labeling, the cells were washed once with KRBB ŽNaCl: 107 mM, KCl: 5 mM, CaCl 2 : 3 mM, MgSO4 : 1 mM, HEPES: 20 mM ŽpH 7.4., glucose: 10 mM, BSA: 0.1%, NaHCO 3 : 7 mM. and pre-incubated with pre-warmed KRBB for 30 min followed by labeling with 32 P phosphoric acid Ž300 mCi. for 3.5 h. At the end of labeling period, the cells were either acutely treated with drugs or lysed directly in lysis buffer ŽHEPES: 50 mM
2. Materials and methods 2.1. Materials Female and male C57BLr6CR mice Ž18–22 g. were purchased from Harlan ŽIndianapolis, IN.. Poly-L-lysine hydrobromide ŽMr ) 300,000., uridine, 5-fluoro-2X-deoxyuridine were purchased from Sigma ŽSt. Louis, MO.. Eagle’s minimum essential medium ŽMEM. and serum were obtained from Gibco ŽSanta Clara, CA.. 32 P phosphoric acid was purchased from NEN Dupont ŽBoston, MA., anti-NMDA ŽR2B. was from Chemicon ŽTemecula, CA., while anti-CsK and anti-Fyn, and anti-mouse IgGcoupled HRPO were obtained from Transduction Laboratories ŽLexington, KY.. All other chemicals were purchased from commercially available sources. 2.2. Cell culture Primary cultures of cerebral cortical neurons were prepared by previously described method w9x. Briefly, cerebral hemispheres were dissected from 15-day old mouse embryos and the cells were separated by trituration using a Pasteur pipette. The separated cells were grown in MEM containing 33.3 mM glucose, 26.2 mM NaHCO3, 100 mM glutamine, 10% heat-inactivated horse serum and 10% fetal bovine serum ŽMEM 10r10. in an incubator with 95% O 2r5% CO 2 at 378C. The plated cultures were incubated for 24 h, after which time 75% of the growth medium was replaced with the same volume of medium containing 10% heat-inactivated horse serum only ŽMEM 10., and a mixture of sterile 5-fluoro-2X-deoxyuridine and uridine at a final concentration of 10 mgrml was added to control the proliferation of non-neuronal cells. After 2 days of culture in vitro, neurons were treated with either 75 mM ethanol or 1 mM MK-801 or a combination of both Ž75 mM ethanolq 1 mM MK-801. for 5 days. The medium and the appropriate
Fig. 1. Effect of acute ethanol treatment on the phosphorylation of the NMDAR2B subunit in mouse cultured cortical neurons. Cortical neurons were cultured for 5 days and labeled with 32 P H 3 PO4 for 3.5 h. The neurons were treated with 75 and 150 mM ethanol for 10 min and the labeled NMDAR2B subunit was immunoprecipitated and transferred to membrane for analysis. Lanes represent: 1s control; 2 s 75 mM ethanol; and 3s150 mM ethanol. The results are summarized in Fig. 2. ŽA. Autoradiogram and ŽB. Western blot.
H.S.G. Kalluri, M.K. Tickur Molecular Brain Research 68 (1999) 159–168
ŽpH 7.4., NaCl: 150 mM, Triton X-100: 1%, Bacitracin: 1 mgrml, B-glycerophosphate: 50 mM, NaF: 10 mM, PMSF: 1 mM, N-ethyl maleimide: 1 mM., and incubated on ice for 1 h followed by centrifugation at 12,000 rpm for 10 min to remove debris. The NMDAR2B antibody Ž1 ml. was added to the cell lysate and incubated at 48C for 1.5 h followed by the addition of 30 ml of Gamma Bind G Sepharose beads and incubation continued for another 1.5 h. The sepharose beads were washed several times, and the proteins eluted from beads by the Laemmli sample buffer w12x. 2.4. Tyrosine phosphorylation of R2B subunit in adult mice Mice were injected with ethanol Ž3.5 grkg. and same volume of saline was administered to control animals. Cerebral cortex and hippocampus were dissected after 5 min of ethanol administration and homogenized in lysis buffer Ž10 mM Tris–HCl ŽpH 7.4., 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, 1 mM sodium ortho-vanadate, 1 mM PMSF, 10 mgrml each of pepstatin, leupeptin. as described w17x and the R2B subunit was immunoprecipitated from both control and ethanol-treated mice as described above. 2.5. Phosphotyrosine detection The protocol followed for phosphotyrosine detection was exactly as above except that the cells were not labeled with 32 P phosphoric acid, but lysed in lysis buffer containing sodium ortho-vanadate.
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2.6. Immunoblotting Membrane proteins or immunoprecipitated protein were separated by SDS–PAGE w12x and transferred to polyvinylidene difluoride membrane w26x. The 32 P labeled protein was detected by exposing X-ray film to the membrane for overnight. The membrane was blocked with 5% milkr0.1% Tween 20 in Tris-buffered saline for 1 h followed by overnight incubation with primary antibody. After several washes, peroxidase-coupled secondary antibody Žeither anti-rabbit or anti-mouse. was added and incubated for 1 h. The membrane was washed and specific bands were visualized using ECL detection kit ŽAmersham, Arlington Heights, IL.. To determine tyrosine phosphorylation, the blot was probed with phosphotyrosine specific antibodies ŽPY20. and then after stripping, reprobed with the R2B antibody to normalize the protein content.
3. Results The effect of ethanol and MK-801 on the phosphorylation of the NMDAR2B subunit in mammalian cultured cortical neurons was examined in the present study. Pilot experiment was performed by using various concentrations of antibody to immunoprecipitate R2B subunit protein. The amount of R2B subunit immunoprecipitated, increased from 0.5 to 1 to 1.5 ml of antibody used. Hence, in further experiments 1 ml of antibody was used for immunoprecip-
Fig. 2. Graphic analysis of the data presented in Fig. 1. The values are mean" S.E.M. of three experiments.
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Fig. 3. Effect of acute and chronic ethanol treatment on the phosphorylation of the NMDAR2B subunit in cortical neurons. The neurons were exposed to 75 mM ethanol for 10 min, 20 min, and 5 days in culture and labeled as described in Section 2. The labeled NMDAR2B subunit was immunoprecipitated and quantitated for phosphorylation. Lanes represent: 1 s control; 2 s 10 min; 3 s 20 min; and 4 s 5 days. ŽA. Autoradiogram and ŽB. Western blot.
itation studies. In vivo labeling with 32 P phosphoric acid demonstrated that acute ethanol Ž75 and 150 mM. treatment did not alter the total phosphorylation of the R2B subunit ŽFigs. 1 and 2.. Treatment of the cortical neuronal cells with 75 mM ethanol for 10 min, 20 min, and 5 days Žchronic. in vitro also did not affect the phosphorylation of the R2B subunit ŽFigs. 3 and 4.. Likewise, chronic treat-
ment of the cortical neurons with the NMDA receptor antagonist MK-801 Ž1 mM; 5 days., did not affect the phosphorylation of the R2B subunit Ždata not shown.. To examine the role of tyrosine phosphorylation of the R2B subunit, phosphotyrosine specific antibodies ŽPY-20. were used to detect any change in the tyrosine containing residues of control, ethanol Ž75 mM., MK-801 Ž1 mM.,
Fig. 4. Graphic representation of the data in Fig. 3. The values represent mean" S.E.M. of three experiments.
H.S.G. Kalluri, M.K. Tickur Molecular Brain Research 68 (1999) 159–168
and ethanol Ž75 mM. q MK-801 Ž1 mM. treated cortical neurons. Immunoblot analysis indicated that tyrosine phosphorylation of the R2B subunit did not change significantly following these treatments ŽFigs. 5 and 6.. To normalize the data, the same blot was stripped and reprobed with the NMDAR2B antibody, results of which show that the same quantity of protein was precipitated during immunoprecipitation protocol. Since cortical neurons do not express R2A subunit at 21 days of development w6,28x, we studied the possibility of the R2B phosphorylation by Fyn kinase. The effect of
163
ethanol and MK-801 on the expression of Fyn kinase and Csk is shown in Figs. 7 and 8. The data indicate that Fyn kinase was present in both 21-day old neuronal cells and also in adult brain, while Csk was present in cortical neurons but absent in adult brain. Further, there was no significant difference in the content of either Fyn kinase or Csk between control, ethanol or MK-801 treated neurons. In vivo ethanol administration Ž3.5 grkg. to mice also did not show any change in the phosphorylation of the R2B subunit in the cerebral cortex of C57BL mice as compared to the control. However, there was an increase
Fig. 5. Tyrosine phosphorylation of the NMDAR2B subunit following ethanol, MK-801, and ethanolq MK-801 treatments. Cortical neurons were cultured in the presence of ethanol Ž75 mM. andror MK-801 Ž1 mM. for 5 days. The neurons were lysed as described in Section 2, and the NMDAR2B subunit was immunoprecipitated, stained once with the phospho-tyrosine antibody ŽPY-20., and reprobed with the R2B antibody to normalize the protein content. Lanes represent: 1 s control; 2 s ethanol; 3 s MK-801; and 4 s ethanolq MK-801. ŽA. PY-20 staining and ŽB. R2B staining.
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Fig. 6. Graphic representation of the data in Fig. 5. The values represent mean" S.E.M. of four experiments.
Fig. 7. Levels of Fyn and Csk kinases in the membranes of ethanol Ž75 mM., MK-801 Ž1 mM., and ethanol Ž75 mM. and MK-801 Ž1 mM. for 5 days. Lanes represent: 1 s control; 2 s ethanol; 3 s MK-801; 4 s ethanolq MK-801, and 5 s adult cerebral cortex. ŽA. Fyn kinase and ŽB. Csk kinase.
H.S.G. Kalluri, M.K. Tickur Molecular Brain Research 68 (1999) 159–168
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Fig. 8. Graphic representation of the data presented in Fig. 7. The values are mean" S.E.M. of four experiments.
Fig. 9. Effect of acute ethanol treatment on the tyrosine phosphorylation of the NMDAR2B subunit in the cerebral cortex and hippocampus of adult C57BL mice. ŽA. Mice were injected 3.5 grkg ethanol and equivalent volume of saline was injected to the control mice. After 5 min, the cerebral cortices and hippocampi were dissected, homogenized in lysis buffer Ž10 mM Tris–HCl ŽpH7.4., 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, 1 mM sodium ortho-vanadate, 1 mM PMSF, 10 mgrml each of pepstatin, leupeptin. and R2B subunit immunoprecipitated and stained as described in Section 2. ŽB. The cortical neurons Ž21-day old. were exposed to 75 mM ethanol for 10 min, lysed in lysis buffer ŽHEPES: 50 mM ŽpH 7.4., NaCl: 150 mM, Triton X-100: 1%, Bacitricin: 1 mgrml, B-glycerophosphate: 50 mM, NaF: 10 mM, PMSF: 1 mM, N-ethyl maleimide: 1 mM. and R2B immunoprecipitated and stained as described in Section 2.
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Fig. 10. Graphic representation of the data presented in Fig. 9. The values are mean " S.E.M. of four individual experiments. U P - 0.05 as compared to control group.
in the tyrosine phosphorylation of the R2B subunit in the hippocampus of ethanol-treated mice ŽFigs. 9 and 10..
4. Discussion Results of our present study show that acute or chronic ethanol treatment did not affect total as well as tyrosine phosphorylation of the NMDA R2B receptor subunit in fetal cortical neurons. Our results also show for the first time that phosphorylation of R2B in these neurons is not altered by NMDA antagonist, MK-801. In a recent study, an up-regulation of tyrosine phosphorylation of the NMDA R2B subunit in hippocampus of adult mice ŽC57BL. was observed following acute ethanol treatment w17x. Miyakawa et al. w17x correlated the observed up-regulation of R2B subunit in hippocampus with Fyn kinase as Fyn deficient homozygous mice did not show enhanced phosphorylation of the R2B subunit. In addition, Fyn deficient mice did not exhibit acute tolerance to ethanol. It should be noted that all the observations reported in this study were related to changes in hippocampal NMDA receptors. A careful examination of their data indicates that Fyn kinase-induced phosphorylation of the R2B subunit occurs in hippocampus. In accordance with the studies of Miyakawa et al.
w17x, we also found that phosphorylation of the R2B subunit in adult C57BL mice in response to acute ethanol was up-regulated only in hippocampus. Further, we observed that acute ethanol treatment of adult mice had no effect on phosphorylation of the R2B subunit in cerebral cortex ŽFig. 9.. Interestingly, acute or chronic ethanol treatment of fetal cortical neurons in vitro did not affect total and tyrosine phosphorylation of the R2B subunit. These results indicate that ethanol induced Fyn kinasemediated phosphorylation of the NMDA R2B subunit occurs in a brain region specific manner. This is an important observation and has physiological and pharmacological consequences. Further studies are required to examine differential regulation of the R2B subunit phosphorylation. Several studies indicate that Src family of tyrosine kinases such as Fyn kinase are negatively regulated via phosphorylation of tyrosine residue at the carboxy terminal by Csk w10,11,18,21x. Inomata et al. w11x demonstrated that the activity of Fyn kinase is inversely proportional to Csk kinase in rat brain. These authors further showed that both Fyn kinase as well as Csk are developmentally regulated in rats. Expression of Fyn kinase begins on E14 Ž14 day embryonic. and reaches the peak level at P35 Žpost-natal. w11x. On the other hand, Csk is expressed at high levels during early development ŽE14-P7. declining gradually
H.S.G. Kalluri, M.K. Tickur Molecular Brain Research 68 (1999) 159–168
and showing little expression at P35 w11x. We have analyzed the expression of Fyn kinase and Csk in murine fetal cortical neurons exposed to ethanol, MK-801 and ethanolrMK-801 by immunoblotting using antibodies against Fyn kinase and Csk. Our results indicate that mouse fetal cortical neurons contain high levels of both Csk and Fyn kinase and that expression of these kinases was not affected by any of the drug treatment ŽFig. 7.. Furthermore, our immunoblot analysis using cerebral cortex of adult mice ŽC57BL. indicated the presence of high levels of Fyn kinase but not the Csk kinase. It is interesting to note that in spite of high abundance of Fyn kinase in adult cerebral cortex, NMDA R2B subunit was not tyrosine phosphorylated in response to acute ethanol ŽFig. 9.. These results suggest that Fyn kinase does not play a role in ethanol-mediated tyrosine phosphorylation of NMDA R2B subunit in cerebral cortex. The phosphorylation of tyrosine residue at the carboxy terminal of Fyn kinase by Csk is pronounced in developing rat brain w11x. It is likely that in our fetal cortical neuronal cultures, Csk negatively regulates Fyn kinase. Our in vitro results obtained using fetal cortical neurons suggest that ethanol-induced, Fyn kinase-mediated tyrosine phosphorylation of R2B subunit may not occur in these neurons. Moreover, based on our results with adult cerebral cortex, it appears that ethanol does not affect the Fyn kinase-mediated phosphorylation of the R2B subunit in this brain region. This is a novel observation and suggests that the R2B subunit of the NMDA receptors can be regulated by ethanol in a brain region specific manner.
w6x
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Acknowledgements w18x
This work was supported in part by National Institute of Health–National Institute on Alcohol Abuse and Alcoholism Grant AA 10552. We thank Drs. A.K. Mehta and M. Kumari for helpful comments, and Ms. Sadie Phillips for excellent secretarial assistance.
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the N-methyl-D-aspartate in mouse cortical neurons in culture, J. Biol. Chem. 271 Ž1996. 13297–13299. P. Follesa, M.K. Ticku, NMDA receptor up-regulation: molecular studies in cultured mouse cortical neurons after chronic antagonist exposure, J. Neurosci. 16 Ž1996. 2172–2178. M. Hollman, S. Heinemann, Cloned glutamate receptors, Annu. Rev. Neurosci. 17 Ž1994. 31–108. G.-Y. Hu, O. Hvalby, S.I. Walaas, K.A. Albert, P. Skjeflo, P. Anderson, P. Greengard, Protein kinase C injection into hippocampal pyramidal cells elicits features of long-term potentiation, Nature 328 Ž1987. 426–429. X.-J. Hu, M.K. Ticku, Chronic ethanol treatment up-regulates the NMDA receptor function and binding in mammalian cortical neurons, Mol. Brain Res. 30 Ž1995. 347–356. A. Imamoto, P. Soriano, Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice, Cell 73 Ž1993. 1117– 1124. M. Inomata, Y. Takayama, H. Kiyama, S. Nada, M. Okada, H. Nakagawa, Regulation of Src family kinases in the developing rat brain: correlation with their regulator kinase, Csk, J. Biol. Chem. 116 Ž1994. 386–392. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 Ž1970. 680–685. L.F. Lau, R.L. Huganir, Differential tyrosine phosphorylation of N-methyl-D-aspartate receptor subunits, J. Biol. Chem. 270 Ž1995. 20036–20041. D.M. Lovinger, G. White, F.F. Weight, Ethanol inhibits NMDAactivated ion channels in hippocampal neurons, Science 243 Ž1989. 1721–1724. H. Markram, M. Segal, Activation of protein kinase C suppresses responses to NMDA in rat CA1 hippocampal neurons, J. Physiol. ŽLond.. 457 Ž1992. 491–501. R.O. Messing, P.J. Petersen, C.J. Henrich, Chronic ethanol exposure increases levels of protein kinase C d and ´ and PKC-mediated phosphorylation in cultured neural cells, J. Biol. Chem. 26 Ž1991. 23428–23432. T. Miyakawa, T. Yagi, H. Kitazawa, M. Yasuda, N. Kawai, K. Tsuboi, H. Niki, Fyn kinase as a determinant of ethanol sensitivity: relation to NMDA receptor function, Science 278 Ž1997. 698–701. S. Nada, T. Yagi, H. Takeda, T. Tokuknaga, H. Nakagawa, Y. Ikawa, M. Okada, S. Aizawa, Constitutive activation of Src family kinases in mouse embryos that lack Csk, Cell 73 Ž1993. 1125–1135. R.A. Nicoll, R.C. Malenka, Contrasting properties of two forms of long-term potentiation in the hippocampus, Nature 377 Ž1995. 115– 118. T.J. O’Dell, E.R. Kandel, S.G.N. Grant, Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors, Nature 353 Ž1991. 558–560. M. Okada, S. Nada, Y. Yamanashi, T. Yamamoto, H. Nakagawa, CSK: a protein-tyrosine kinase involved in regulation of src family kinases, J. Biol. Chem. 266 Ž1991. 24249–24252. K.W. Roche, W.G. Tingley, R.L. Huganir, Glutamate receptor phosphorylation and synaptic plasticity, Curr. Opin. Neurobiol. 4 Ž1994. 383–388. P.H. Seeburg, The molecular biology of the mammalian glutamate receptor channels, Trends Pharmacol. Sci. 14 Ž1993. 297–303. D.L. Snell, B. Tabakoff, P.L. Hoffman, Involvement of protein kinase C in ethanol-induced inhibition of NMDA receptor function in cerebellar granule cells, Alcohol. Clin. Exp. Res. 18 Ž1994. 81–85. W.G. Tingley, K.W. Roche, A.K. Thompson, R.L. Huganir, Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain, Nature 364 Ž1993. 70–73. H.T. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. 76 Ž1979. 4350–4354.
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w27x L. Trevisan, L.W. Fitzgerald, N. Brose, G.P. Gasic, S.F. Heinemann, R.S. Duman, E.J. Nestler, Chronic ingestion of ethanol up-regulates NMDAR1 receptor subunit immunoreactivity in rat hippocampus, J. Neurochem. 62 Ž1994. 1635–1638. w28x K. Williams, M.A. Dichter, P.B. Molinoff, Up-regulation of N-
methyl-D-aspartate receptor on cultured cortical neurons after exposure to antagonists, Mol. Pharmacol. 42 Ž1992. 147–151. w29x X.-M. Yu, R. Askalan, G.J. Keil II, W.S. Michael, NMDA channel regulation by channel-associated protein tyrosine kinase Src, Science 275 Ž1997. 674–677.
Research report
Effect of ethanol on phosphorylation of the NMDAR2B subunit in mouse cortical neurons Haviryaji S.G. Kalluri, Maharaj K. Ticku
)
Department of Pharmacology, The UniÕersity of Texas Health Science Center, 7703 Floyd Curl DriÕe, San Antonio, TX 78284-7764, USA Accepted 2 February 1999
Abstract There is evidence that phosphorylation plays a crucial role in the regulation of the NMDA receptors in the brain. In this study, we examined the effect of acute and chronic ethanol treatment on the phosphorylation of the R2B subunit of the NMDA receptors in fetal cortical neurons. Additionally, the effect of acute ethanol treatment on the phosphorylation of the R2B subunit in adult cerebral cortex and hippocampus was also examined. The results show that acute or chronic ethanol treatments did not affect the total phosphorylation of the R2B subunit in cortical neurons. In adult mice, we observed that acute ethanol treatment increased the tyrosine phosphorylation of the R2B subunit in hippocampus but not in cerebral cortex. We also observed that acute or chronic ethanol treatments did not alter the Fyn or Csk levels in cortical neurons. Although Fyn, but not Csk, was present in adult cerebral cortex, ethanol did not phosphorylate the R2B subunit in this region. Like ethanol, MK-801 ŽNMDA antagonist. did not affect the phosphorylation of the R2B subunit in cortical neurons. Taken together, these results suggest that acute and chronic ethanol and MK-801 treatments do not affect the phosphorylation of the R2B subunit in fetal cortical neurons and adult cerebral cortex. Based on these observations, we speculate that the R2B subunit of the NMDA receptors is regulated by multiple cascades and in a brain region specific manner. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Cultured neuron; NMDA receptor regulation; Phosphorylation; Ethanol; Fyn kinase; Csk kinase
1. Introduction The glutamate receptors mediate fast excitatory transmission in the CNS and play an important role in synaptic plasticity, neuronal development and neurological disorders w3,7,19,23x. Reversible protein phosphorylation by a number of kinases and phosphatases is a major post-translational modification, which regulates a variety of receptor proteins and their function including glutamate receptors w22,29x. Phorbol esters have been shown to increase w1x or decrease w15x NMDA receptor function. Biochemical studies have demonstrated that the NMDA receptors are phosphorylated by PKC and protein tyrosine kinases w13,25x. Earlier studies have shown that long-term potentiation ŽLTP. was blocked by tyrosine kinase inhibitors w20x, and ) Corresponding author. Fax: q1-210-567-4226; E-mail: [email protected]
that LTP-like features were elicited by injecting PKC into hippocampal neurons w8,15x. Since ethanol affects the NMDA receptor function following its acute as well as chronic administration w4– 6,9,14,27x, and that the NMDA receptor function is affected by phosphorylation, it is of interest to study whether ethanol affects the phosphorylation of the NMDA receptors. A previous study has implicated an involvement of PKC in the ethanol-induced inhibition of the NMDA responses w24x. In another study, it was shown that chronic ethanol treatment up-regulates the PKC isozymes Ž d and ´ . and PKC-mediated phosphorylation in neuronal cultures w16x. A recent study has revealed that tyrosine phosphorylation of the NMDAR2B receptor subunit was up-regulated following ethanol treatment in the hippocampus of C57BL mice and this was shown to be mediated by Fyn kinase w17x. These investigators reported that acute tolerance to ethanol in hippocampus involves tyrosine phospho-
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 0 5 7 - 1
160
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rylation of the NMDA receptors by Fyn kinase w17x. Several laboratories have observed a selective and differential up-regulation of the NMDA receptor binding, function, gene, and protein expression in vivo and in vitro w2,4–6,9,27x. Additionally, chronic NMDA receptor antagonists like DŽy.-2-amino-phosphonopentanoic acid ŽAP-5. and MK-801, have been shown to produce up-regulation of NMDA receptor subunit gene and protein expression, an effect similar to that observed following chronic ethanol treatment w6x. In the present study, we investigated the effect of ethanol and MK-801 on the phosphorylation of the NMDAR2B subunit in cultured mouse cortical neurons.
drugs were replaced every 24 h. For acute studies, after 7 days in culture, neurons were treated with 75 or 150 mM for 10 or 20 min, as described below. 2.3. In ÕiÕo labeling and immunoprecipitation Prior to in vivo labeling, the cells were washed once with KRBB ŽNaCl: 107 mM, KCl: 5 mM, CaCl 2 : 3 mM, MgSO4 : 1 mM, HEPES: 20 mM ŽpH 7.4., glucose: 10 mM, BSA: 0.1%, NaHCO 3 : 7 mM. and pre-incubated with pre-warmed KRBB for 30 min followed by labeling with 32 P phosphoric acid Ž300 mCi. for 3.5 h. At the end of labeling period, the cells were either acutely treated with drugs or lysed directly in lysis buffer ŽHEPES: 50 mM
2. Materials and methods 2.1. Materials Female and male C57BLr6CR mice Ž18–22 g. were purchased from Harlan ŽIndianapolis, IN.. Poly-L-lysine hydrobromide ŽMr ) 300,000., uridine, 5-fluoro-2X-deoxyuridine were purchased from Sigma ŽSt. Louis, MO.. Eagle’s minimum essential medium ŽMEM. and serum were obtained from Gibco ŽSanta Clara, CA.. 32 P phosphoric acid was purchased from NEN Dupont ŽBoston, MA., anti-NMDA ŽR2B. was from Chemicon ŽTemecula, CA., while anti-CsK and anti-Fyn, and anti-mouse IgGcoupled HRPO were obtained from Transduction Laboratories ŽLexington, KY.. All other chemicals were purchased from commercially available sources. 2.2. Cell culture Primary cultures of cerebral cortical neurons were prepared by previously described method w9x. Briefly, cerebral hemispheres were dissected from 15-day old mouse embryos and the cells were separated by trituration using a Pasteur pipette. The separated cells were grown in MEM containing 33.3 mM glucose, 26.2 mM NaHCO3, 100 mM glutamine, 10% heat-inactivated horse serum and 10% fetal bovine serum ŽMEM 10r10. in an incubator with 95% O 2r5% CO 2 at 378C. The plated cultures were incubated for 24 h, after which time 75% of the growth medium was replaced with the same volume of medium containing 10% heat-inactivated horse serum only ŽMEM 10., and a mixture of sterile 5-fluoro-2X-deoxyuridine and uridine at a final concentration of 10 mgrml was added to control the proliferation of non-neuronal cells. After 2 days of culture in vitro, neurons were treated with either 75 mM ethanol or 1 mM MK-801 or a combination of both Ž75 mM ethanolq 1 mM MK-801. for 5 days. The medium and the appropriate
Fig. 1. Effect of acute ethanol treatment on the phosphorylation of the NMDAR2B subunit in mouse cultured cortical neurons. Cortical neurons were cultured for 5 days and labeled with 32 P H 3 PO4 for 3.5 h. The neurons were treated with 75 and 150 mM ethanol for 10 min and the labeled NMDAR2B subunit was immunoprecipitated and transferred to membrane for analysis. Lanes represent: 1s control; 2 s 75 mM ethanol; and 3s150 mM ethanol. The results are summarized in Fig. 2. ŽA. Autoradiogram and ŽB. Western blot.
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ŽpH 7.4., NaCl: 150 mM, Triton X-100: 1%, Bacitracin: 1 mgrml, B-glycerophosphate: 50 mM, NaF: 10 mM, PMSF: 1 mM, N-ethyl maleimide: 1 mM., and incubated on ice for 1 h followed by centrifugation at 12,000 rpm for 10 min to remove debris. The NMDAR2B antibody Ž1 ml. was added to the cell lysate and incubated at 48C for 1.5 h followed by the addition of 30 ml of Gamma Bind G Sepharose beads and incubation continued for another 1.5 h. The sepharose beads were washed several times, and the proteins eluted from beads by the Laemmli sample buffer w12x. 2.4. Tyrosine phosphorylation of R2B subunit in adult mice Mice were injected with ethanol Ž3.5 grkg. and same volume of saline was administered to control animals. Cerebral cortex and hippocampus were dissected after 5 min of ethanol administration and homogenized in lysis buffer Ž10 mM Tris–HCl ŽpH 7.4., 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, 1 mM sodium ortho-vanadate, 1 mM PMSF, 10 mgrml each of pepstatin, leupeptin. as described w17x and the R2B subunit was immunoprecipitated from both control and ethanol-treated mice as described above. 2.5. Phosphotyrosine detection The protocol followed for phosphotyrosine detection was exactly as above except that the cells were not labeled with 32 P phosphoric acid, but lysed in lysis buffer containing sodium ortho-vanadate.
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2.6. Immunoblotting Membrane proteins or immunoprecipitated protein were separated by SDS–PAGE w12x and transferred to polyvinylidene difluoride membrane w26x. The 32 P labeled protein was detected by exposing X-ray film to the membrane for overnight. The membrane was blocked with 5% milkr0.1% Tween 20 in Tris-buffered saline for 1 h followed by overnight incubation with primary antibody. After several washes, peroxidase-coupled secondary antibody Žeither anti-rabbit or anti-mouse. was added and incubated for 1 h. The membrane was washed and specific bands were visualized using ECL detection kit ŽAmersham, Arlington Heights, IL.. To determine tyrosine phosphorylation, the blot was probed with phosphotyrosine specific antibodies ŽPY20. and then after stripping, reprobed with the R2B antibody to normalize the protein content.
3. Results The effect of ethanol and MK-801 on the phosphorylation of the NMDAR2B subunit in mammalian cultured cortical neurons was examined in the present study. Pilot experiment was performed by using various concentrations of antibody to immunoprecipitate R2B subunit protein. The amount of R2B subunit immunoprecipitated, increased from 0.5 to 1 to 1.5 ml of antibody used. Hence, in further experiments 1 ml of antibody was used for immunoprecip-
Fig. 2. Graphic analysis of the data presented in Fig. 1. The values are mean" S.E.M. of three experiments.
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Fig. 3. Effect of acute and chronic ethanol treatment on the phosphorylation of the NMDAR2B subunit in cortical neurons. The neurons were exposed to 75 mM ethanol for 10 min, 20 min, and 5 days in culture and labeled as described in Section 2. The labeled NMDAR2B subunit was immunoprecipitated and quantitated for phosphorylation. Lanes represent: 1 s control; 2 s 10 min; 3 s 20 min; and 4 s 5 days. ŽA. Autoradiogram and ŽB. Western blot.
itation studies. In vivo labeling with 32 P phosphoric acid demonstrated that acute ethanol Ž75 and 150 mM. treatment did not alter the total phosphorylation of the R2B subunit ŽFigs. 1 and 2.. Treatment of the cortical neuronal cells with 75 mM ethanol for 10 min, 20 min, and 5 days Žchronic. in vitro also did not affect the phosphorylation of the R2B subunit ŽFigs. 3 and 4.. Likewise, chronic treat-
ment of the cortical neurons with the NMDA receptor antagonist MK-801 Ž1 mM; 5 days., did not affect the phosphorylation of the R2B subunit Ždata not shown.. To examine the role of tyrosine phosphorylation of the R2B subunit, phosphotyrosine specific antibodies ŽPY-20. were used to detect any change in the tyrosine containing residues of control, ethanol Ž75 mM., MK-801 Ž1 mM.,
Fig. 4. Graphic representation of the data in Fig. 3. The values represent mean" S.E.M. of three experiments.
H.S.G. Kalluri, M.K. Tickur Molecular Brain Research 68 (1999) 159–168
and ethanol Ž75 mM. q MK-801 Ž1 mM. treated cortical neurons. Immunoblot analysis indicated that tyrosine phosphorylation of the R2B subunit did not change significantly following these treatments ŽFigs. 5 and 6.. To normalize the data, the same blot was stripped and reprobed with the NMDAR2B antibody, results of which show that the same quantity of protein was precipitated during immunoprecipitation protocol. Since cortical neurons do not express R2A subunit at 21 days of development w6,28x, we studied the possibility of the R2B phosphorylation by Fyn kinase. The effect of
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ethanol and MK-801 on the expression of Fyn kinase and Csk is shown in Figs. 7 and 8. The data indicate that Fyn kinase was present in both 21-day old neuronal cells and also in adult brain, while Csk was present in cortical neurons but absent in adult brain. Further, there was no significant difference in the content of either Fyn kinase or Csk between control, ethanol or MK-801 treated neurons. In vivo ethanol administration Ž3.5 grkg. to mice also did not show any change in the phosphorylation of the R2B subunit in the cerebral cortex of C57BL mice as compared to the control. However, there was an increase
Fig. 5. Tyrosine phosphorylation of the NMDAR2B subunit following ethanol, MK-801, and ethanolq MK-801 treatments. Cortical neurons were cultured in the presence of ethanol Ž75 mM. andror MK-801 Ž1 mM. for 5 days. The neurons were lysed as described in Section 2, and the NMDAR2B subunit was immunoprecipitated, stained once with the phospho-tyrosine antibody ŽPY-20., and reprobed with the R2B antibody to normalize the protein content. Lanes represent: 1 s control; 2 s ethanol; 3 s MK-801; and 4 s ethanolq MK-801. ŽA. PY-20 staining and ŽB. R2B staining.
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Fig. 6. Graphic representation of the data in Fig. 5. The values represent mean" S.E.M. of four experiments.
Fig. 7. Levels of Fyn and Csk kinases in the membranes of ethanol Ž75 mM., MK-801 Ž1 mM., and ethanol Ž75 mM. and MK-801 Ž1 mM. for 5 days. Lanes represent: 1 s control; 2 s ethanol; 3 s MK-801; 4 s ethanolq MK-801, and 5 s adult cerebral cortex. ŽA. Fyn kinase and ŽB. Csk kinase.
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Fig. 8. Graphic representation of the data presented in Fig. 7. The values are mean" S.E.M. of four experiments.
Fig. 9. Effect of acute ethanol treatment on the tyrosine phosphorylation of the NMDAR2B subunit in the cerebral cortex and hippocampus of adult C57BL mice. ŽA. Mice were injected 3.5 grkg ethanol and equivalent volume of saline was injected to the control mice. After 5 min, the cerebral cortices and hippocampi were dissected, homogenized in lysis buffer Ž10 mM Tris–HCl ŽpH7.4., 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, 1 mM sodium ortho-vanadate, 1 mM PMSF, 10 mgrml each of pepstatin, leupeptin. and R2B subunit immunoprecipitated and stained as described in Section 2. ŽB. The cortical neurons Ž21-day old. were exposed to 75 mM ethanol for 10 min, lysed in lysis buffer ŽHEPES: 50 mM ŽpH 7.4., NaCl: 150 mM, Triton X-100: 1%, Bacitricin: 1 mgrml, B-glycerophosphate: 50 mM, NaF: 10 mM, PMSF: 1 mM, N-ethyl maleimide: 1 mM. and R2B immunoprecipitated and stained as described in Section 2.
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Fig. 10. Graphic representation of the data presented in Fig. 9. The values are mean " S.E.M. of four individual experiments. U P - 0.05 as compared to control group.
in the tyrosine phosphorylation of the R2B subunit in the hippocampus of ethanol-treated mice ŽFigs. 9 and 10..
4. Discussion Results of our present study show that acute or chronic ethanol treatment did not affect total as well as tyrosine phosphorylation of the NMDA R2B receptor subunit in fetal cortical neurons. Our results also show for the first time that phosphorylation of R2B in these neurons is not altered by NMDA antagonist, MK-801. In a recent study, an up-regulation of tyrosine phosphorylation of the NMDA R2B subunit in hippocampus of adult mice ŽC57BL. was observed following acute ethanol treatment w17x. Miyakawa et al. w17x correlated the observed up-regulation of R2B subunit in hippocampus with Fyn kinase as Fyn deficient homozygous mice did not show enhanced phosphorylation of the R2B subunit. In addition, Fyn deficient mice did not exhibit acute tolerance to ethanol. It should be noted that all the observations reported in this study were related to changes in hippocampal NMDA receptors. A careful examination of their data indicates that Fyn kinase-induced phosphorylation of the R2B subunit occurs in hippocampus. In accordance with the studies of Miyakawa et al.
w17x, we also found that phosphorylation of the R2B subunit in adult C57BL mice in response to acute ethanol was up-regulated only in hippocampus. Further, we observed that acute ethanol treatment of adult mice had no effect on phosphorylation of the R2B subunit in cerebral cortex ŽFig. 9.. Interestingly, acute or chronic ethanol treatment of fetal cortical neurons in vitro did not affect total and tyrosine phosphorylation of the R2B subunit. These results indicate that ethanol induced Fyn kinasemediated phosphorylation of the NMDA R2B subunit occurs in a brain region specific manner. This is an important observation and has physiological and pharmacological consequences. Further studies are required to examine differential regulation of the R2B subunit phosphorylation. Several studies indicate that Src family of tyrosine kinases such as Fyn kinase are negatively regulated via phosphorylation of tyrosine residue at the carboxy terminal by Csk w10,11,18,21x. Inomata et al. w11x demonstrated that the activity of Fyn kinase is inversely proportional to Csk kinase in rat brain. These authors further showed that both Fyn kinase as well as Csk are developmentally regulated in rats. Expression of Fyn kinase begins on E14 Ž14 day embryonic. and reaches the peak level at P35 Žpost-natal. w11x. On the other hand, Csk is expressed at high levels during early development ŽE14-P7. declining gradually
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and showing little expression at P35 w11x. We have analyzed the expression of Fyn kinase and Csk in murine fetal cortical neurons exposed to ethanol, MK-801 and ethanolrMK-801 by immunoblotting using antibodies against Fyn kinase and Csk. Our results indicate that mouse fetal cortical neurons contain high levels of both Csk and Fyn kinase and that expression of these kinases was not affected by any of the drug treatment ŽFig. 7.. Furthermore, our immunoblot analysis using cerebral cortex of adult mice ŽC57BL. indicated the presence of high levels of Fyn kinase but not the Csk kinase. It is interesting to note that in spite of high abundance of Fyn kinase in adult cerebral cortex, NMDA R2B subunit was not tyrosine phosphorylated in response to acute ethanol ŽFig. 9.. These results suggest that Fyn kinase does not play a role in ethanol-mediated tyrosine phosphorylation of NMDA R2B subunit in cerebral cortex. The phosphorylation of tyrosine residue at the carboxy terminal of Fyn kinase by Csk is pronounced in developing rat brain w11x. It is likely that in our fetal cortical neuronal cultures, Csk negatively regulates Fyn kinase. Our in vitro results obtained using fetal cortical neurons suggest that ethanol-induced, Fyn kinase-mediated tyrosine phosphorylation of R2B subunit may not occur in these neurons. Moreover, based on our results with adult cerebral cortex, it appears that ethanol does not affect the Fyn kinase-mediated phosphorylation of the R2B subunit in this brain region. This is a novel observation and suggests that the R2B subunit of the NMDA receptors can be regulated by ethanol in a brain region specific manner.
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This work was supported in part by National Institute of Health–National Institute on Alcohol Abuse and Alcoholism Grant AA 10552. We thank Drs. A.K. Mehta and M. Kumari for helpful comments, and Ms. Sadie Phillips for excellent secretarial assistance.
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