Neurochemical Research

, Volume 15, Issue 10, pp 1023–1029 | Cite as

Characterization of ouabain-induced phosphoinositide hydrolysis in brain slices of the neonatal rat

  • Walter Balduini
  • Lucio G. Costa
Original Articles


The effect of the Na/K-ATPase inhibitor ouabain on phosphoinositide (Ptdlns) hydrolysis was studied in rat brain cortical slices. Ouabain induced a dose-dependent accumulation of inositol phosphates (InsPs) which was much higher in neonatal rats (1570±40% of basal) than in adult animals (287±18% of basal). For this reason, all experiments were conducted with 7 day-old rats. Strophantidin caused a similar stimulation of Ptdlns hydrolysis, although it was less potent than ouabain. The order of potency for ouabain-stimulated InsPs accumulation in brain areas was hippocampus>cortex>brainstem>cerebellum. The effect of ouabain was not blocked by antagonists for the muscarinic, alpha1-adrenergic and glutamate receptors. Also ineffective were the K+ channel blockers 4-aminopyridine and tetraethylammonium, the sodium channel blocker tetrodotoxin, and the calcium channel blocker verapamil, whereas the Na/Ca exchanger blocker amiloride partially antagonized the effect of ouabain. The accumulation of InsPs induced by ouabain was additive to that of carbachol and norepinephrine, as well as to that induced by high K+ and veratrine, but not to that of glutamate. Removal of Na+ ions from the incubation buffer completely prevented the accumulation of InsPs induced by ouabain. The effect of ouabain was also dependent upon extracellular calcium and was under negative feedback control of protein kinase C. Despite the higher effect of ouabain on Ptdlns hydrolysis of immature rats, the density of [3H]ouabain binding sites, as well as the activity of Na/K-ATPase were higher in adult animals. Furthermore, a poor correlation was found between ouabain-stimulated Ptdlns hydrolysis and [3H]ouabain binding in brain regions. These results suggest an involvement of Na+ pump in the hydrolysis of Ptdlns, possibly related to an effect on Na+ and Ca2+ homeostasis. The immature rat appear to be an useful model for studying the relationship between Na/K-ATPase and inositol metabolism.

Key Words

Ouabain Na/K-ATPase phosphoinositide hydrolysis 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Abdel-Latif, A. A. 1986. Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Pharmacol. Rev. 38:227–272.PubMedGoogle Scholar
  2. 2.
    Berridge, M. J. 1984. Inositol triphosphate and diacylglycerol: two interacting second messengers. Ann. Rev. Biochem. 56:159–193.Google Scholar
  3. 3.
    Hirasawa, K., and Nishizuka, Y. 1985. Phosphatidylinositol turnover in receptor mechanisms and signal transduction. Ann. Rev. Pharmacol. Toxicol. 25:147–170.Google Scholar
  4. 4.
    Gusovski, F. Hollingsworth, E. B., and Daly, J. W. 1986. Regulation of phosphatidylinositol turnover in brain synaptoneurosomes: stimulatory effects of agents that enhance influx of sodium ions. Proc. Natl. Acad. Sci. USA 83:3003–3007.PubMedGoogle Scholar
  5. 5.
    Gusovski, F., McNeal, E., and Daly, J. W. 1987. Stimulation of phosphoinositide breakdown in brain synaptoneurosomes by agents that activate sodium influx: antagonism by tetrodotoxin, saxitoxin, and cadmium. Mol. Pharmacol. 32:479–487.PubMedGoogle Scholar
  6. 6.
    Gusovski, F., and Daly, J. W. 1988. Formation of inositol phosphates in synaptoneurosomes of guinea-pig brain: stimulatory effects of receptor agonists, sodium channel agents and sodium and calcium ionophores. Neuropharmacol. 27:95–105.Google Scholar
  7. 7.
    Court, J. A., Fowler, C. J., Candy, J. M., Hoban, P. R., and Smith, C. J. 1986. Raising the ambient potassium ion concentration enhances carbachol stimulated phosphoinositide hydrolysis in rat brain hippocampal and cerebral cortical miniprism. Naunyn-Schmiedeberg's Arch. Pharmacol. 334:10–16.Google Scholar
  8. 8.
    Habermann, H., and Laux, M. 1986. Depolarization increases inositolphosphate production in a particulate preparation from rat brain. Naunyn-Schmiedeberg's Arch. Pharmacol. 334:1–9.Google Scholar
  9. 9.
    Osborne, N. N. 1988. Tricyclic antidepressants, mianserin, and ouabain stimulate inositol phosphate formation in vitro in rat cortical slices. Neurochem. Res. 13:105–111.PubMedGoogle Scholar
  10. 10.
    Diamant, S., and Daphne, A. 1989. Potentiation of [3H]inositol phosphate formation by receptor activation and membrane depolarization in brain cortical slices. Brain Res. 503:55–61.PubMedGoogle Scholar
  11. 11.
    Balduini, W., Murphy, S. D., and Costa, L. G. 1987. Developmental changes in muscarinic receptor-stimulated phosphoinositide metabolism in rat brain. J. Pharmacol. Exp. Ther. 241:421–427.PubMedGoogle Scholar
  12. 12.
    Balduini, W., Costa, L. G., and Murphy, S. D. 1990. Potassium ions potentiate the muscarinic receptor-stimulated phosphoinositide metabolism in cerebral cortex slices: a comparison of neonatal and adult rats. Neurochem. Res. 15:33–39.PubMedGoogle Scholar
  13. 13.
    Berridge, M. J. Downes, C. P., and Hanley, M. R. 1982. Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem. J. 206:587–595.PubMedGoogle Scholar
  14. 14.
    Costa, L. G., Kaylor, G., and Murphy, S. D. 1986. Carbacholand norepinephrine-stimulated phosphoinositide metabolism in rat brain: effect of chronic cholinesterase inhibition. J. Pharmacol. Exp. Ther. 239:32–37.PubMedGoogle Scholar
  15. 15.
    Brown, E., Kendall, D. A., and Nahorski, S. R. 1984. Inositol phospholipid hydrolysis in rat cerebral cortex slices. I. Receptor characterization. J. Neurochem. 42:1379–1387.PubMedGoogle Scholar
  16. 16.
    Hauger, R., Luu, M. D., Meyer, D. K., Goodwin, F. K., and Paul, S. M. 1985. Characterization of “High-Affinity” [3H]Ouabain binding in the rat central nervous system. J. Neurochem. 44:1709–1715.PubMedGoogle Scholar
  17. 17.
    Phillips, T. D., and Hayes, A. W. 1977. Effects of patulin on adenosine triphosphatase activities in the mouse. Toxicol. Appl. Pharmacol. 42:175–187.PubMedGoogle Scholar
  18. 18.
    Costa, L. G. 1985. Inhibition of [3H]Aminobutyric acid uptake by organotin compounds in vivo. Toxicol. Appl. Pharmacol. 79:471–479.PubMedGoogle Scholar
  19. 19.
    Taussky, H. H., and Shorr, E. 1953. A microcolorimetric method for the determination of inorganic phosphorus. J. Biol. Chem. 202:675–685.PubMedGoogle Scholar
  20. 20.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.PubMedGoogle Scholar
  21. 21.
    Kendall, D. A., and Nahorski, S. R. 1984. Inositol phospholipid hydrolysis in rat cerebral cortical slices: calcium requirement. J. Neurochem. 42:1388–1394.PubMedGoogle Scholar
  22. 22.
    Labarca, R., Janowsky, A., Patel, J., and Paul, S. M. 1984. Phorbol esters inhibit agonist-induced [3H]inositol-1-phosphate accumulation in rat hippocampal slices. Biochem. Biophys. Res. Commun. 123:703–709.PubMedGoogle Scholar
  23. 23.
    Balduini, W., Murphy, S. D., and Costa, L. G. 1990. Characterization of cholinergic muscarinic receptor-stimulated phosphoinositide metabolism in brain from immature rats. J. Pharmacol. Exp. Ther. 253:573–579.PubMedGoogle Scholar
  24. 24.
    Stahl, W. L. 1986. The Na, K-ATPase of the nervous tissue. Neurochem. Int. 8:449–476.Google Scholar
  25. 25.
    Anner, B. M. 1985. The receptor function of the Na+, K+-activated adenosine triphosphate system. Biochem. J. 227:1–11.PubMedGoogle Scholar
  26. 26.
    Kendall, D. A., and Nahorski, S. R. 1987. Depolarization-evoked release of acetylcholine can mediate phosphoinositide hydrolysis in slices of rat cerebral cortex. Neuropharmacol. 26:513–519.Google Scholar
  27. 27.
    Benuck, M., Reith, M. E. A., and Lajtha, A. 1989. Phosphoinositide hydrolysis induced by depolarization and sodium channel activation in mouse cerebralcortical slices. Neuropharmacol. 28:847–854.Google Scholar
  28. 28.
    Stahl, W. L., and Swanson, P. D. 1972. Calcium movements in brain slices in low sodium and calcium medium. J. Neurochem. 19:2395–2407.PubMedGoogle Scholar
  29. 29.
    Smith, J. B., Dwyer, S. D., and Smith, L. 1989. Decreasing extracellular Na+ concentration triggers inositol polyphosphate production and Ca2+ mobilization. J. Biol. Chem. 264:831–837.PubMedGoogle Scholar
  30. 30.
    Sasaguri, T., and Watson, S. P. 1988. Lowering of the extracellular Na+ concentration enhances high K+-induced formation of inositol phosphates in the guinea-pig ileum. Biochem. J. 252:883–888.PubMedGoogle Scholar
  31. 31.
    Blaustein, M. P. 1988. Calcium trapped and buffering in neurons. Trends Neurosci. 11:438–443.PubMedGoogle Scholar
  32. 32.
    Coutinho, O. P., Carvalho, C. A. M., and Carvalho, A. P. 1984. Calcium uptake related to K+-depolarization and Na+/Ca2+ exchange in sheep brain synaptosomes. Brain Res. 290:261–271.PubMedGoogle Scholar
  33. 33.
    Wilson, D. B., Bross, T. E., Hofmann, S. L., and Majerus, P. W. 1984. Hydrolysis of phosphoinositides by purified sheep seminal vesicle phospholipase C enzyme. J. Biol. Chem. 259:11718–11724.PubMedGoogle Scholar
  34. 34.
    Samson, F. E., and Quinn, D. J. 1967. Na+-K+-activated ATPase in rat brain development. J. Neurochem. 14:421–427.PubMedGoogle Scholar
  35. 35.
    Nicoletti, F., Iadarola, M. J., Wroblewski, J. T., and Costa, E. 1986. Excitatory aminoacid recognition sites coupled with inositol phospholipid metabolism: developomental changes and interaction with alpha1-adrenoreceptors. Proc. Natl. Acad. Sci. USA 83:1931–1935.PubMedGoogle Scholar
  36. 36.
    Simmons, D. A., Kern, F. O., Winecard, A. I., and Martin, D. B. 1986. Basal phsophatidilinositol turnover controls aortic Na+/K+ATPase activity. J. Clin. Invest. 77:242–245.Google Scholar
  37. 37.
    Greene, D. A. and Lattimer, S. A. 1986. Protein kinase C agonists acutely normalize decreased ouabain-inhibitable respiration in diabetic rabbit nerve. Diabets 35:242–245.Google Scholar
  38. 38.
    Lattimer, S. A., Sima, A. A. F., and Greene, D. A. 1989. In vitro correction of impaired Na+/K+-ATPase in diabetic nerve by proteik kinase C agonists. Am. J. Physiol. 256:E264-E269.PubMedGoogle Scholar
  39. 39.
    Schellenberg, G. D., and Swanson, 1981. Sodium-dependent and calcium-dependent calcium transport by rat brain microsomes. Biochem. Biophis. Acta 648:13–27.Google Scholar
  40. 40.
    Gill, D. L., Grollman, E. F., and Kolhn L. D. 1984. Calcium transport mechanisms in membrane vescicles guinea-pig brain synaptosomes. J. Biol. Chem. 259:11718–11724.PubMedGoogle Scholar
  41. 41.
    Scheleenberg, G. D., Anderson, L., and Swanson, P. D. 1983. Inhibition of Na+-Ca+ exchange in rat brain by amiloride. Mol. Pharmacol. 24:251–258.PubMedGoogle Scholar
  42. 42.
    Dobbing, J., and Sands, J. 1979. Comparative aspects of the brain growth spurt. Early Human Dev. 3:79–83.Google Scholar

Copyright information

© Plenum Publishing Corporation 1990

Authors and Affiliations

  • Walter Balduini
    • 1
  • Lucio G. Costa
    • 1
  1. 1.Department of Environmental Health, SC-34University of WashingtonSeattle

Personalised recommendations