Cellular and Molecular Neurobiology

, Volume 29, Issue 1, pp 69–80 | Cite as

Altered Pattern of Na,K-ATPase Activity and mRNA During Chronic Alcohol Consumption by Juvenile and Adolescent Rats

Original Paper


The effect of chronic ethanol consumption on cerebral cortical activity of Na,K-ATPase was determined in Long-Evans (LE) rats fed an ethanol-containing diet beginning at different stages of development. Na,K-ATPase activity was operationally resolved into α1 and α2/3 isozyme activities. There was no significant difference in Na,K-ATPase activities before and after alcohol consumption in the preparations from adult rats. However, for rats beginning alcohol consumption as adolescents, the α2/3 activity was significantly elevated following chronic alcohol consumption. Both LE and Sprague–Dawley rats showed this same selective increase in cortical α2/3 activity when rats began alcohol consumption as juveniles. The shift in cortical α2/3 activity was not observed in cerebellum or subcortical forebrain and was reversible when rats were fed ethanol throughout the normal adolescent period and then withdrawn and tested 2 weeks later (during the adult period). Levels of isoform-specific mRNA were determined in preparations of cerebral cortices of rats showing elevated α2/3 isozyme activities. In these preparations, isoform specific α2 and α3 mRNA was significantly elevated. There was no effect of ethanol feeding on cortical α1 mRNA. These findings indicate that the longer term effects of ethanol on the developing brain include elevated Na,K-ATPase activity and a mechanism that is pre-translational and isoform specific.


Adolescence Brain development Chronic ethanol consumption mRNA levels Na K-ATPase Long-Evans rat 



Early portions of this work were supported by PHS grant AA07848 and by a grant-in-aid from the URI Council for Research. The authors thanks Dr. Jerry B. Lingrel for providing the isozyme specific probes, Dr. Joel Chandlee for providing lab space and advice on Northern blot analysis, Dr. Paul Cohen for providing E. coli HB101, Dr. Stanley Bower for providing M13/pUC primers, and Dr. Brian Maynard for advice and use of the NIH image analysis program for slot blot analysis. The authors acknowledge the assistance of Ms. Jane Knapp and Dr. Jiongdong Pang with the Northern blot analysis and the help of Sean Casavant, Philip Empey, Joe Susa, and Bruno Soffientino (URI) in administering the liquid diets and caring for the animals. Jennifer Huggan (MU) assisted in BAC determinations.


  1. Akera T, Rech RH, Marquis WJ, Tobin T, Brody TM (1973) Lack of relationship between brain (Na + K)-activated adenosine triphosphatase and the development of tolerance to ethanol in rats. J Pharmacol Exp Ther 185:594–601PubMedGoogle Scholar
  2. Aloia RC, Paxton J, Daviau JS, Gelb OV, Mlekusch W, Truppe W et al (1985) Effect of chronic alcohol consumption on rat brain microsome lipid composition, membrane fluidity and Na,K-ATPase activity. Life Sci 36:1003–1017. doi: 10.1016/0024-3205(85)90398-4 PubMedCrossRefGoogle Scholar
  3. Baginsk ES, Foa PP, Zak B (1967) Microdetermination of inorganic phosphate, phospholipids, and total phosphate in biologic materials. Clin Chem 13:326–332Google Scholar
  4. Barada K, Okolo C, Field M, Cortas N (1994) Na,K-ATPase in diabetic rat intestine. Changes at protein and mRNA levels and role of glucagon. J Clin Invest 93:2725–2731. doi: 10.1172/JCI117287 PubMedCrossRefGoogle Scholar
  5. Beauge F, Stibler H, Kalant H (1983) Brain synaptosomal Na,K-ATPase activity as an index of tolerance to ethanol. Pharmacol Biochem Behav 18(suppl):519–524. doi: 10.1016/0091-3057(83)90229-0 PubMedCrossRefGoogle Scholar
  6. Bertoni JM, Sprenkle PM (1994) Effects of in vitro ethanol on the brain cation pump in alcoholics and controls. Biochem Pharmacol 48:435–437. doi: 10.1016/0006-2952(94)90120-1 PubMedCrossRefGoogle Scholar
  7. Blaustein MP (1988) Calcium transport and buffering in neurons. Trends Neurosci 11:438–443. doi: 10.1016/0166-2236(88)90195-6 PubMedCrossRefGoogle Scholar
  8. Brodsky JL (1990) Insulin activation of brain Na,K-ATPase is mediated by α2-form of enzyme. Am J Physiol 258:C812–C817PubMedGoogle Scholar
  9. Brodsky JL, Guidotti G (1990) Sodium affinity of brain Na,K-ATPase is dependent on isozyme and environment of the pump. Am J Physiol 258:C803–C811PubMedGoogle Scholar
  10. Charlemagne D, Orlowski J, Oliviero P, Rannou F, Sainte Beuve C, Swynghedauw B et al (1994) Alteration of Na,K-ATPase subunit mRNA and protein levels in hypertrophied rat heart. J Biol Chem 269:1541–1547PubMedGoogle Scholar
  11. Chen Y, Wixom PM, Sun AY (1997) Enhanced (Na + K)ATPase activity and expression in mouse brain after chronic ethanol administration. Neurochem Res 22:583–588. doi: 10.1023/A:1022470019604 PubMedCrossRefGoogle Scholar
  12. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. doi: 10.1016/0003-2697(87)90021-2 PubMedCrossRefGoogle Scholar
  13. Chung C-S, Wang J, Wehman M, Rhoads DE (2008) Severity of alcohol withdrawal symptoms depends on developmental stage of Long-Evans rats. Pharmacol Biochem Behav 89:137–144. doi: 10.1016/j.pbb.2007.12.002 PubMedCrossRefGoogle Scholar
  14. Cicero TJ, Adams ML, O’Connor L, Nock B, Meyer ER, Wozniak D (1990) Influence of chronic alcohol administration of representative indices of puberty and sexual maturation in male rats and the development of their progeny. J Pharmacol Exp Ther 255:707–715PubMedGoogle Scholar
  15. Crews F, He J, Hodge C (2007) Adolescent cortical development: a critical period of vulnerability for addiction. Pharmacol Biochem Behav 86:189–199. doi: 10.1016/j.pbb.2006.12.001 PubMedCrossRefGoogle Scholar
  16. Doremus TL, Brunell SC, Rajendran P, Spear LP (2005) Factors influencing elevated ethanol consumption in adolescent relative to adult rats. Alcohol Clin Exp Res 29:1796–1808. doi: 10.1097/01.alc.0000183007.65998.aa PubMedCrossRefGoogle Scholar
  17. Foley TD, Rhoads DE (1992) Effects of ethanol on Na-dependent amino acid uptake: depencence on rat age and Na,K-ATPase activity. Brain Res 593:39–44. doi: 10.1016/0006-8993(92)91260-L PubMedCrossRefGoogle Scholar
  18. Foley TD, Rhoads DE (1994) Stimulation of synaptosomal Na,K-ATPase by ethanol: possible involvement of an isozyme-specific inhibitor of Na,K-ATPase. Brain Res 653:167–172. doi: 10.1016/0006-8993(94)90386-7 PubMedCrossRefGoogle Scholar
  19. Foster DM, Huber MD, Klemm WR (1989) Ethanol may stimulate or inhibit Na,K-ATPase, depending upon Na and K concentrations. Alcohol 6:437–443. doi: 10.1016/0741-8329(89)90048-7 PubMedCrossRefGoogle Scholar
  20. Goldstein DB, Israel Y (1972) Effects of ethanol on mouse brain microsomal (Na + K)-activated adenosine triphosphatase. Life Sci II 11:957–963PubMedGoogle Scholar
  21. Gray EG, Whittaker VP (1962) The isolation of nerve endings from brain: an electron microscopic study of cell fragments derived by homogenization and centrifugation. J Anat 96:79–88PubMedGoogle Scholar
  22. Guerri C, Grisolia S (1983) Chronic ethanol treatment affects synaptosomal membrane-bound enzymes. Pharmacol Biochem Behav 18(suppl):45–50. doi: 10.1016/0091-3057(83)90145-4 PubMedCrossRefGoogle Scholar
  23. Herrera VL, Cova T, Sassoon D, Ruiz-Opazo N (1994) Developmental cell-specific regulation of Na,K-ATPase alpha 1-, alpha 2-, and alpha 3-isoform gene expression. Am J Physiol 266:C1301–C1312PubMedGoogle Scholar
  24. Ikeda K, Onaka T, Yamakado M, Nakai J, Ishikawa TO, Taketo MM et al (2003) Degeneration of the amygdala/piriform cortex and enhanced fear/anxiety behaviors in sodium pump alpha 2 subunit (Atp1a2)-deficient mice. J Neurosci 23:4667–4676PubMedGoogle Scholar
  25. Israel Y, Kalant H, LeBlanc AE, Bernstein JC, Salazar I (1970) Changes in cation transport and (Na + K)-activated adenosine triphosphatase produced by chronic administration of ethanol. J Pharmacol Exp Ther 174:330–336PubMedGoogle Scholar
  26. Juhaszova M, Blaustein MP (1997) Na+ pump low and high ouabain affinity alpha subunit isoforms are differentially distributed in cells. Proc Natl Acad Sci USA 94:1800–1805. doi: 10.1073/pnas.94.5.1800 PubMedCrossRefGoogle Scholar
  27. Leventhal M, Tabakoff B (1980) Sodium-potassium activated adenosine triphosphatase activity as a measure of neuronal membrane characteristics in ethanol-tolerant mice. J Pharmacol Exp Ther 212:315–319Google Scholar
  28. Lieber CS, DeCarli LM (1982) The feeding of alcohol in liquid diets: two decades of applications and 1982 update. Alcohol Clin Exp Res 6:523–531. doi: 10.1111/j.1530-0277.1982.tb05017.x PubMedCrossRefGoogle Scholar
  29. Majchrowicz E (1975) Induction of physical dependence upon ethanol and the associated behavioral changes in rats. Psychopharmacology (Berl) 43:245–2454. doi: 10.1007/BF00429258 CrossRefGoogle Scholar
  30. Majchrowicz E (1981) Reversal in central nervous system function during ethanol withdrawal in humans and experimental animals. Fed Proc 40:2065–2072PubMedGoogle Scholar
  31. Marks MJ, Smolen A, Collins AC (1984) Brain Na,K-ATPase in mice differentially sensitive to alcohols. Alcohol Clin Exp Res 8:390–396. doi: 10.1111/j.1530-0277.1984.tb05685.x PubMedCrossRefGoogle Scholar
  32. Markwiese BJ, Acheson SK, Levin ED, Wilson WA, Swartzwelder HS (1998) Differential effects of ethanol on memory in adolescent and adult rats. Alcohol Clin Exp Res 22:416–421PubMedGoogle Scholar
  33. McGrail KM, Phillips JM, Sweadner KJ (1991) Immunofluorescent localization of three Na,K-ATPase isozymes in the rat central nervous system: both neurons and glia can express more than one Na,K-ATPase. J Neurosci 11:381–391PubMedGoogle Scholar
  34. Moseley AE, Lieske SP, Wetzel RK, James PF, He S, Shelly DA et al (2003) The Na,K-ATPase alpha 2 isoform is expressed in neurons, and its absence disrupts neuronal activity in newborn mice. J Biol Chem 278:5317–5324. doi: 10.1074/jbc.M211315200 PubMedCrossRefGoogle Scholar
  35. Moseley AE, Williams MT, Schaefer TL, Bohanan CS, Neumann JC, Behbehani MM et al (2007) Deficiency in Na,K-ATPase alpha isoform genes alters spatial learning, motor activity, and anxiety in mice. J Neurosci 27:616–626. doi: 10.1523/JNEUROSCI.4464-06.2007 PubMedCrossRefGoogle Scholar
  36. Nhamburo PT, Salafsky BP, Tabakoff B, Hoffman PL (1987) Effects of ethanol on ouabain inhibition of mouse brain Na,K-ATPase activity. Biochem Pharmacol 36:2027–2033. doi: 10.1016/0006-2952(87)90504-1 PubMedCrossRefGoogle Scholar
  37. Orlowski J, Lingrel JB (1988) Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic α isoform and α subunit mRNAs. J Biol Chem 263:10436–10442PubMedGoogle Scholar
  38. Rabin RA (1988) Different response of adenylate cyclase and ATPase activities after chronic ethanol exposure of PC12 cells. J Neurochem 51:1148–1152. doi: 10.1111/j.1471-4159.1988.tb03080.x PubMedCrossRefGoogle Scholar
  39. Rabin RA, Acara MA (1993) Regulation of Na,K-ATPase by chronic ethanol exposure of PC12 cells. Biochem Pharmacol 45:1652–1658. doi: 10.1016/0006-2952(93)90306-H CrossRefGoogle Scholar
  40. Rangaraj N, Kalant H (1984) Effects of ethanol tolerance on norepinephrine-ethanol inhibition of Na,K-adenosine triphosphatase in various regions of rat brain. J Pharmacol Exp Ther 231:416–421PubMedGoogle Scholar
  41. Rao PA, Kumari CL, Sadasivudu B (1985) Acute and short term effects of ethanol on membrane enzymes in rat brain. Neurochem Res 10:1577–1585. doi: 10.1007/BF00988600 PubMedCrossRefGoogle Scholar
  42. Rhoads DE, Peterson NA, Sankaran H, Raghupathy E (1982) Inhibitory effects of scorpion venom on the uptake of amino acids by synaptosomes and synaptosomal memmbranes. Biochem Pharmacol 31:1875–1879. doi: 10.1016/0006-2952(82)90490-7 PubMedCrossRefGoogle Scholar
  43. Shull MM, Lingrel JB (1987) Multiple genes encode the human Na,K-ATPase catalytic subunit. Proc Natl Acad Sci USA 84:4039–4043. doi: 10.1073/pnas.84.12.4039 PubMedCrossRefGoogle Scholar
  44. Shull GE, Greeb J, Lingrel JB (1986) Molecular cloning of three distinct forms of the Na,K-ATPase α subunit from rat brain. Biochem 25:8125–8132. doi: 10.1021/bi00373a001 CrossRefGoogle Scholar
  45. Spear LP (2000a) The adolescent brain and age-related behavioral manifestations. Neurosci Biobehav Rev 24:417–463. doi: 10.1016/S0149-7634(00)00014-2 PubMedCrossRefGoogle Scholar
  46. Spear LP (2000b) Modeling adolescent development and alcohol use in animals. Alcohol Res Health 24:115–123PubMedGoogle Scholar
  47. Spear LP, Varlinskaya EI (2005) Adolescence: alcohol sensitivity, tolerance and intake. Recent Dev Alcohol 17:143–159. doi: 10.1007/0-306-48626-1_7 PubMedCrossRefGoogle Scholar
  48. Sun AY, Samorajski T (1970) Effect of ethanol on the activity of adenosine triphosphatase and acetylcholinesterase in synaptosomes isolated from guinea pig brain. J Neurochem 17:1365–1372. doi: 10.1111/j.1471-4159.1970.tb06871.x PubMedCrossRefGoogle Scholar
  49. Swann AC (1990) Ethanol inhibition of active 86Rb(+)-transport: evidence for enhancement by sodium or calcium influx. J Pharmacol Exp Ther 254:864–871PubMedGoogle Scholar
  50. Swartzwelder HS, Wilson WA, Tayyeb MI (1995) Differential sensitivity of NMDA receptor-mediated synaptic potentials to ethanol in immature verse mature hippocampus. Alcohol Clin Exp Res 19:320–323. doi: 10.1111/j.1530-0277.1995.tb01509.x PubMedCrossRefGoogle Scholar
  51. Sweadner KJ (1989) Isozymes of the Na,K-ATPase. Biochim Biophys Acta 988:185–220PubMedGoogle Scholar
  52. Sweadner KJ (1992) Overlapping and diverse distribution of Na,K-ATPase isozymes in neurons and glia. Can J Physiol Pharmacol 70(Suppl):S255–S259PubMedGoogle Scholar
  53. Urayama O, Nakao M (1979) Organ specificity of rat sodium- and potassium-activated adenosine triphosphatase. J Biochem 86:1371–1381PubMedGoogle Scholar
  54. Vetter CS, Doremus-Fitzwater TL, Spear LP (2007) Time course of elevated ethanol intake in adolescent relative to adult rats under continuous, voluntary-access conditions. Alcohol Clin Exp Res 31:1159–1168. doi: 10.1111/j.1530-0277.2007.00417.x PubMedCrossRefGoogle Scholar
  55. Wang J, Adachi M, Rhoads DE (1998) A calnaktin-like inhibitor of Na,K-ATPase in rat brain: regulation of α1 and α2 isozymes. Comp Biochem Physiol B Biochem Mol Biol 119:241–246. doi: 10.1016/S0305-0491(98)01002-5 PubMedCrossRefGoogle Scholar
  56. White AM, Swartzwelder HS (2005) Age-related effects of alcohol on memory and memory-related brain funcion in adolescents and adults. Res Dev Alcohol 17:161–176. doi: 10.1007/0-306-48626-1_8 CrossRefGoogle Scholar
  57. Witt ED (1994) Mechanisms of alcohol abuse and alcoholism in adolescents: a case for developing animal models. Behav Neural Biol 62:168–177. doi: 10.1016/S0163-1047(05)80015-9 PubMedCrossRefGoogle Scholar
  58. Young RM, Lingrel JB (1987) Tissue distribution of mRNAs encoding the isoforms and subunit of rat Na,K-ATPase. Biochem Biophys Res Commun 145:52–58. doi: 10.1016/0006-291X(87)91286-1 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Jian Wang
    • 1
  • Chun-Shiang Chung
    • 1
  • Dennis E. Rhoads
    • 1
    • 2
  1. 1.Department of Biochemistry, Microbiology and Molecular GeneticsUniversity of Rhode IslandKingstonUSA
  2. 2.Department of BiologyMonmouth UniversityNJUSA

Personalised recommendations