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. 1985 Jul 1;101(1):269–276. doi: 10.1083/jcb.101.1.269

Osmotic and phorbol ester-induced activation of Na+/H+ exchange: possible role of protein phosphorylation in lymphocyte volume regulation

PMCID: PMC2113623  PMID: 2989300

Abstract

The Na+/H+ antiport is stimulated by 12-O-tetradecanoylphorbol-13, acetate (TPA) and other phorbol esters in rat thymic lymphocytes. Mediation by protein kinase C is suggested by three findings: (a) 1- oleoyl-2-acetylglycerol also activated the antiport; (b) trifluoperazine, an inhibitor of protein kinase C, blocked the stimulation of Na+/H+ exchange; and (c) activation of countertransport was accompanied by increased phosphorylation of specific membrane proteins. The Na+/H+ antiport is also activated by osmotic cell shrinking. The time course, extent, and reversibility of the osmotically induced and phorbol ester-induced responses are similar. Moreover, the responses are not additive and they are equally susceptible to inhibition by trifluoperazine, N-ethylmaleimide, and ATP depletion. The extensive analogies between the TPA and osmotically induced effects suggested a common underlying mechanism, possibly activation of a protein kinase. It is conceivable that osmotic shrinkage initiates the following sequence of events: stimulation of protein kinase(s) followed by activation of the Na+/H+ antiport, resulting in cytoplasmic alkalinization. The Na+ taken up through the antiport, together with the HCO3- and Cl- accumulated in the cells as a result of the cytoplasmic alkalinization, would be followed by osmotically obliged water. This series of events could underlie the phenomenon of regulatory volume increase.

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Selected References

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  1. Aronson P. S. Mechanisms of active H+ secretion in the proximal tubule. Am J Physiol. 1983 Dec;245(6):F647–F659. doi: 10.1152/ajprenal.1983.245.6.F647. [DOI] [PubMed] [Google Scholar]
  2. Aronson P. S., Nee J., Suhm M. A. Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature. 1982 Sep 9;299(5879):161–163. doi: 10.1038/299161a0. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J. Inositol trisphosphate and diacylglycerol as second messengers. Biochem J. 1984 Jun 1;220(2):345–360. doi: 10.1042/bj2200345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Besterman J. M., Cuatrecasas P. Phorbol esters rapidly stimulate amiloride-sensitive Na+/H+ exchange in a human leukemic cell line. J Cell Biol. 1984 Jul;99(1 Pt 1):340–343. doi: 10.1083/jcb.99.1.340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Cala P. M. Volume regulation by Amphiuma red blood cells. The membrane potential and its implications regarding the nature of the ion-flux pathways. J Gen Physiol. 1980 Dec;76(6):683–708. doi: 10.1085/jgp.76.6.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cassel D., Whiteley B., Zhuang Y. X., Glaser L. Mitogen-independent activation of Na+/H+ exchange in human epidermoid carcinoma A431 cells: regulation by medium osmolarity. J Cell Physiol. 1985 Feb;122(2):178–186. doi: 10.1002/jcp.1041220203. [DOI] [PubMed] [Google Scholar]
  8. Grinstein S., Cohen S., Goetz J. D., Rothstein A., Gelfand E. W. Characterization of the activation of Na+/H+ exchange in lymphocytes by phorbol esters: change in cytoplasmic pH dependence of the antiport. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1429–1433. doi: 10.1073/pnas.82.5.1429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grinstein S., Cohen S., Rothstein A. Chemical modification of the Na+/H+ exchanger of thymic lymphocytes. Inhibition by N-ethylmaleimide. Biochim Biophys Acta. 1985 Jan 10;812(1):213–222. doi: 10.1016/0005-2736(85)90541-3. [DOI] [PubMed] [Google Scholar]
  10. Grinstein S., Cohen S., Rothstein A. Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H+ antiport. J Gen Physiol. 1984 Mar;83(3):341–369. doi: 10.1085/jgp.83.3.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grinstein S., Goetz J. D., Furuya W., Rothstein A., Gelfand E. W. Amiloride-sensitive Na+-H+ exchange in platelets and leukocytes: detection by electronic cell sizing. Am J Physiol. 1984 Sep;247(3 Pt 1):C293–C298. doi: 10.1152/ajpcell.1984.247.3.C293. [DOI] [PubMed] [Google Scholar]
  12. Grinstein S., Goetz J. D., Rothstein A. 22Na+ fluxes in thymic lymphocytes. II. Amiloride-sensitive Na+/H+ exchange pathway; reversibility of transport and asymmetry of the modifier site. J Gen Physiol. 1984 Oct;84(4):585–600. doi: 10.1085/jgp.84.4.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kraft A. S., Anderson W. B. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature. 1983 Feb 17;301(5901):621–623. doi: 10.1038/301621a0. [DOI] [PubMed] [Google Scholar]
  14. Kuo J. F., Andersson R. G., Wise B. C., Mackerlova L., Salomonsson I., Brackett N. L., Katoh N., Shoji M., Wrenn R. W. Calcium-dependent protein kinase: widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoperazine. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7039–7043. doi: 10.1073/pnas.77.12.7039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Moolenaar W. H., Tertoolen L. G., de Laat S. W. Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH. Nature. 1984 Nov 22;312(5992):371–374. doi: 10.1038/312371a0. [DOI] [PubMed] [Google Scholar]
  16. Moolenaar W. H., Tsien R. Y., van der Saag P. T., de Laat S. W. Na+/H+ exchange and cytoplasmic pH in the action of growth factors in human fibroblasts. Nature. 1983 Aug 18;304(5927):645–648. doi: 10.1038/304645a0. [DOI] [PubMed] [Google Scholar]
  17. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  18. Paris S., Pouysségur J. Biochemical characterization of the amiloride-sensitive Na+/H+ antiport in Chinese hamster lung fibroblasts. J Biol Chem. 1983 Mar 25;258(6):3503–3508. [PubMed] [Google Scholar]
  19. Parker J. C., Castranova V. Volume-responsive sodium and proton movements in dog red blood cells. J Gen Physiol. 1984 Sep;84(3):379–401. doi: 10.1085/jgp.84.3.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rink R. J., Sanchez A., Grinstein S., Rothstein A. Volume restoration in osmotically swollen lymphocytes does not involve changes in free Ca2+ concentration. Biochim Biophys Acta. 1983 Jul 14;762(4):593–596. doi: 10.1016/0167-4889(83)90064-2. [DOI] [PubMed] [Google Scholar]
  21. Rosoff P. M., Stein L. F., Cantley L. C. Phorbol esters induce differentiation in a pre-B-lymphocyte cell line by enhancing Na+/H+ exchange. J Biol Chem. 1984 Jun 10;259(11):7056–7060. [PubMed] [Google Scholar]
  22. Rothenberg P., Glaser L., Schlesinger P., Cassel D. Activation of Na+/H+ exchange by epidermal growth factor elevates intracellular pH in A431 cells. J Biol Chem. 1983 Oct 25;258(20):12644–12653. [PubMed] [Google Scholar]
  23. Simpson A. W., Hallam T. J., Rink T. J. TMB-8 inhibits secretion evoked by phorbol ester at basal cytoplasmic free calcium in quin2-loaded platelets much more effectively than it inhibits thrombin-induced calcium mobilisation. FEBS Lett. 1984 Oct 15;176(1):139–143. doi: 10.1016/0014-5793(84)80928-x. [DOI] [PubMed] [Google Scholar]
  24. Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]
  25. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Weinman S. A., Reuss L. Na+-H+ exchange at the apical membrane of Necturus gallbladder. Extracellular and intracellular pH studies. J Gen Physiol. 1982 Aug;80(2):299–321. doi: 10.1085/jgp.80.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Whiteley B., Cassel D., Zhuang Y. X., Glaser L. Tumor promoter phorbol 12-myristate 13-acetate inhibits mitogen-stimulated Na+/H+ exchange in human epidermoid carcinoma A431 cells. J Cell Biol. 1984 Sep;99(3):1162–1166. doi: 10.1083/jcb.99.3.1162. [DOI] [PMC free article] [PubMed] [Google Scholar]

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