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. 1990 Aug 1;269(3):757–766. doi: 10.1042/bj2690757

Micromolar free calcium exposes ouabain-binding sites in digitonin-permeabilized Xenopus laevis oocytes.

G Schmalzing 1, S Kröner 1
PMCID: PMC1131652  PMID: 2167664

Abstract

As demonstrated previously, digitonin-permeabilized Xenopus oocytes have a large internal pool of sodium pumps which are inaccessible to cytosolic ouabain [Schmalzing, Kröner & Passow (1989) Biochem. J. 260, 395-399]. Access to internal ouabain-binding sites required permeabilization of inner membranes with SDS. In the present study, micromolar free Ca2+ was found to stimulate ouabain binding in the digitonin-permeabilized cells (K0.5 0.5 microM-Ca2+, h 1.9, average of seven experiments) without disrupting intracellular membranes. Sustained incubation at 9 microM-Ca2+ was as effective as SDS in inducing access to the ouabain-binding sites of the internal sodium pumps. Omission of either Mg2+ or ATP completely abolished the Ca2+ effect. Half-maximal stimulation by Ca2+ required approx. 0.4 mM-MgATP. Of a variety of nucleotides tested, none was as effective as ATP (rank order ATP greater than ADP greater than ATP[S] (adenosine 5'-[gamma-thio]triphosphate) greater than CTP greater than UTP greater than ITP = XTP greater than GTP). Pi, AMP, cyclic AMP, cyclic GMP, GTP[S] (guanosine 5'-[gamma-thio]triphosphate) and a stable ATP analogue p[NH]ppA (adenosine 5'-[beta gamma-imido]triphosphate), were ineffective. The metalloendoproteinase inhibitor carbobenzoxy-Gly-Phe-amide reduced the Ca2+ effect by some 50%. Inhibitors of chymotrypsin and the Ca2+ proteinase calpain had no effect. Ca2+ ionophores (A23187 and ionomycin) and the polycations neomycin and polymixin B blocked the Ca2+ response entirely. Neomycin also abolished a Ca2(+)-independent stimulation of ouabain binding by the wasp venom mastoparan. The requirements for increasing the accessibility of ouabain-binding sites are remarkably similar to those for exocytosis in secretory cells, suggesting that oocytes and eggs possess a Ca2(+)-regulated pathway for the plasma membrane insertion of sodium pumps.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ammann D., Bührer T., Schefer U., Müller M., Simon W. Intracellular neutral carrier-based Ca2+ microelectrode with subnanomolar detection limit. Pflugers Arch. 1987 Jul;409(3):223–228. doi: 10.1007/BF00583469. [DOI] [PubMed] [Google Scholar]
  2. Atkinson K. D., Ramirez R. M. Secretion can proceed uncoupled from net plasma membrane expansion in inositol-starved Saccharomyces cerevisiae. J Bacteriol. 1984 Oct;160(1):80–86. doi: 10.1128/jb.160.1.80-86.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baker P. F., Knight D. E. Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Nature. 1978 Dec 7;276(5688):620–622. doi: 10.1038/276620a0. [DOI] [PubMed] [Google Scholar]
  4. Baker P. F., Whitaker M. J. Influence of ATP and calcium on the cortical reaction in sea urchin eggs. Nature. 1978 Nov 30;276(5687):513–515. doi: 10.1038/276513a0. [DOI] [PubMed] [Google Scholar]
  5. Brown D. Vesicle recycling and cell-specific function in kidney epithelial cells. Annu Rev Physiol. 1989;51:771–784. doi: 10.1146/annurev.ph.51.030189.004011. [DOI] [PubMed] [Google Scholar]
  6. Burgess T. L., Kelly R. B. Constitutive and regulated secretion of proteins. Annu Rev Cell Biol. 1987;3:243–293. doi: 10.1146/annurev.cb.03.110187.001331. [DOI] [PubMed] [Google Scholar]
  7. Busa W. B., Nuccitelli R. An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis. J Cell Biol. 1985 Apr;100(4):1325–1329. doi: 10.1083/jcb.100.4.1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cannon C., van Adelsberg J., Kelly S., Al-Awqati Q. Carbon-dioxide-induced exocytotic insertion of H+ pumps in turtle-bladder luminal membrane: role of cell pH and calcium. Nature. 1985 Apr 4;314(6010):443–446. doi: 10.1038/314443a0. [DOI] [PubMed] [Google Scholar]
  9. Cockcroft S., Gomperts B. D. Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase. Nature. 1985 Apr 11;314(6011):534–536. doi: 10.1038/314534a0. [DOI] [PubMed] [Google Scholar]
  10. Couch C. B., Strittmatter W. J. Specific blockers of myoblast fusion inhibit a soluble and not the membrane-associated metalloendoprotease in myoblasts. J Biol Chem. 1984 May 10;259(9):5396–5399. [PubMed] [Google Scholar]
  11. Dumont J. N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J Morphol. 1972 Feb;136(2):153–179. doi: 10.1002/jmor.1051360203. [DOI] [PubMed] [Google Scholar]
  12. Dunn L. A., Holz R. W. Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells. J Biol Chem. 1983 Apr 25;258(8):4989–4993. [PubMed] [Google Scholar]
  13. Farach H. A., Jr, Mundy D. I., Strittmatter W. J., Lennarz W. J. Evidence for the involvement of metalloendoproteases in the acrosome reaction in sea urchin sperm. J Biol Chem. 1987 Apr 25;262(12):5483–5487. [PubMed] [Google Scholar]
  14. Gomperts B. D. Involvement of guanine nucleotide-binding protein in the gating of Ca2+ by receptors. Nature. 1983 Nov 3;306(5938):64–66. doi: 10.1038/306064a0. [DOI] [PubMed] [Google Scholar]
  15. Hansen O. Facilitation of ouabain binding to (Na+ + K+)-ATPase by vanadate at in vivo concentrations. Biochim Biophys Acta. 1979 May 10;568(1):265–269. doi: 10.1016/0005-2744(79)90293-6. [DOI] [PubMed] [Google Scholar]
  16. Harris B., Cheek T. R., Burgoyne R. D. Effects of metalloendoproteinase inhibitors on secretion and intracellular free calcium in bovine adrenal chromaffin cells. Biochim Biophys Acta. 1986 Oct 31;889(1):1–5. doi: 10.1016/0167-4889(86)90002-9. [DOI] [PubMed] [Google Scholar]
  17. Haslam R. J., Davidson M. M. Guanine nucleotides decrease the free [Ca2+] required for secretion of serotonin from permeabilized blood platelets. Evidence of a role for a GTP-binding protein in platelet activation. FEBS Lett. 1984 Aug 20;174(1):90–95. doi: 10.1016/0014-5793(84)81084-4. [DOI] [PubMed] [Google Scholar]
  18. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984 Oct 9;23(21):5036–5041. doi: 10.1021/bi00316a032. [DOI] [PubMed] [Google Scholar]
  19. Holz R. W., Bittner M. A., Peppers S. C., Senter R. A., Eberhard D. A. MgATP-independent and MgATP-dependent exocytosis. Evidence that MgATP primes adrenal chromaffin cells to undergo exocytosis. J Biol Chem. 1989 Apr 5;264(10):5412–5419. [PubMed] [Google Scholar]
  20. Jochen A. L., Berhanu P. Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes. J Cell Biol. 1986 Nov;103(5):1807–1816. doi: 10.1083/jcb.103.5.1807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kenigsberg R. L., Trifaró J. M. Microinjection of calmodulin antibodies into cultured chromaffin cells blocks catecholamine release in response to stimulation. Neuroscience. 1985 Jan;14(1):335–347. doi: 10.1016/0306-4522(85)90183-6. [DOI] [PubMed] [Google Scholar]
  22. Kimura T., Imamura K., Eckhardt L., Schulz I. Ca2+-, phorbol ester-, and cAMP-stimulated enzyme secretion from permeabilized rat pancreatic acini. Am J Physiol. 1986 May;250(5 Pt 1):G698–G708. doi: 10.1152/ajpgi.1986.250.5.G698. [DOI] [PubMed] [Google Scholar]
  23. Knight D. E., Baker P. F. Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields. J Membr Biol. 1982;68(2):107–140. doi: 10.1007/BF01872259. [DOI] [PubMed] [Google Scholar]
  24. Knight D. E., Sugden D., Baker P. F. Evidence implicating protein kinase C in exocytosis from electropermeabilized bovine chromaffin cells. J Membr Biol. 1988 Aug;104(1):21–34. doi: 10.1007/BF01871899. [DOI] [PubMed] [Google Scholar]
  25. Lienhard G. E., Secemski I. I. P 1 ,P 5 -Di(adenosine-5')pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. J Biol Chem. 1973 Feb 10;248(3):1121–1123. [PubMed] [Google Scholar]
  26. Lodhi S., Weiner N. D., Schacht J. Interactions of neomycin and calcium in synaptosomal membranes and polyphosphoinostide monolayers. Biochim Biophys Acta. 1976 Apr 5;426(4):781–785. doi: 10.1016/0005-2736(76)90147-4. [DOI] [PubMed] [Google Scholar]
  27. McLaughlin S., Whitaker M. Cations that alter surface potentials of lipid bilayers increase the calcium requirement for exocytosis in sea urchin eggs. J Physiol. 1988 Feb;396:189–204. doi: 10.1113/jphysiol.1988.sp016958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Missiaen L., Wuytack F., Raeymaekers L., De Smedt H., Casteels R. Polyamines and neomycin inhibit the purified plasma-membrane Ca2+ pump by interacting with associated polyphosphoinositides. Biochem J. 1989 Aug 1;261(3):1055–1058. doi: 10.1042/bj2611055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mundy D. I., Strittmatter W. J. Requirement for metalloendoprotease in exocytosis: evidence in mast cells and adrenal chromaffin cells. Cell. 1985 Mar;40(3):645–656. doi: 10.1016/0092-8674(85)90213-2. [DOI] [PubMed] [Google Scholar]
  30. Nakajima T., Uzu S., Wakamatsu K., Saito K., Miyazawa T., Yasuhara T., Tsukamoto Y., Fujino M. Amphiphilic peptides in wasp venom. Biopolymers. 1986;25 (Suppl):S115–S121. [PubMed] [Google Scholar]
  31. Okano Y., Takagi H., Tohmatsu T., Nakashima S., Kuroda Y., Saito K., Nozawa Y. A wasp venom mastoparan-induced polyphosphoinositide breakdown in rat peritoneal mast cells. FEBS Lett. 1985 Sep 2;188(2):363–366. doi: 10.1016/0014-5793(85)80403-8. [DOI] [PubMed] [Google Scholar]
  32. Poenie M., Alderton J., Tsien R. Y., Steinhardt R. A. Changes of free calcium levels with stages of the cell division cycle. Nature. 1985 May 9;315(6015):147–149. doi: 10.1038/315147a0. [DOI] [PubMed] [Google Scholar]
  33. Richter H. P., Jung D., Passow H. Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation of Xenopus oocytes. J Membr Biol. 1984;79(3):203–210. doi: 10.1007/BF01871059. [DOI] [PubMed] [Google Scholar]
  34. Ronning S. A., Martin T. F. Characterization of Ca2+-stimulated secretion in permeable GH3 pituitary cells. J Biol Chem. 1986 Jun 15;261(17):7834–7839. [PubMed] [Google Scholar]
  35. Sarafian T., Aunis D., Bader M. F. Loss of proteins from digitonin-permeabilized adrenal chromaffin cells essential for exocytosis. J Biol Chem. 1987 Dec 5;262(34):16671–16676. [PubMed] [Google Scholar]
  36. Schmalzing G., Eckard P., Kröner S., Passow H. Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes. Am J Physiol. 1990 Jan;258(1 Pt 1):C179–C184. doi: 10.1152/ajpcell.1990.258.1.C179. [DOI] [PubMed] [Google Scholar]
  37. Schmalzing G., Kröner S., Passow H. Evidence for intracellular sodium pumps in permeabilized Xenopus laevis oocytes. Biochem J. 1989 Jun 1;260(2):395–399. doi: 10.1042/bj2600395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schwartz G. J., Al-Awqati Q. Regulation of transepithelial H+ transport by exocytosis and endocytosis. Annu Rev Physiol. 1986;48:153–161. doi: 10.1146/annurev.ph.48.030186.001101. [DOI] [PubMed] [Google Scholar]
  39. Steinhardt R. A., Alderton J. M. Calmodulin confers calcium sensitivity on secretory exocytosis. Nature. 1982 Jan 14;295(5845):154–155. doi: 10.1038/295154a0. [DOI] [PubMed] [Google Scholar]
  40. Strous G. J., van Kerkhof P., Dekker J., Schwartz A. L. Metalloendoprotease inhibitors block protein synthesis, intracellular transport, and endocytosis in hepatoma cells. J Biol Chem. 1988 Dec 5;263(34):18197–18204. [PubMed] [Google Scholar]
  41. Tadolini B., Varani E. Interaction of spermine with polyphosphoinositides containing liposomes and myo-inositol 1,4,5 triphosphate. Biochem Biophys Res Commun. 1986 Feb 26;135(1):58–64. doi: 10.1016/0006-291x(86)90942-3. [DOI] [PubMed] [Google Scholar]
  42. TerBush D. R., Holz R. W. Effects of phorbol esters, diglyceride, and cholinergic agonists on the subcellular distribution of protein kinase C in intact or digitonin-permeabilized adrenal chromaffin cells. J Biol Chem. 1986 Dec 25;261(36):17099–17106. [PubMed] [Google Scholar]
  43. Vacquier V. D. The isolation of intact cortical granules from sea urchin eggs: calcium lons trigger granule discharge. Dev Biol. 1975 Mar;43(1):62–74. doi: 10.1016/0012-1606(75)90131-1. [DOI] [PubMed] [Google Scholar]
  44. Wade J. B. Role of membrane fusion in hormonal regulation of epithelial transport. Annu Rev Physiol. 1986;48:213–223. doi: 10.1146/annurev.ph.48.030186.001241. [DOI] [PubMed] [Google Scholar]
  45. Whitaker M., Aitchison M. Calcium-dependent polyphosphoinositide hydrolysis is associated with exocytosis in vitro. FEBS Lett. 1985 Mar 11;182(1):119–124. doi: 10.1016/0014-5793(85)81167-4. [DOI] [PubMed] [Google Scholar]
  46. Wilson S. P. Effects of mastoparan on catecholamine release from chromaffin cells. FEBS Lett. 1989 Apr 24;247(2):239–241. doi: 10.1016/0014-5793(89)81343-2. [DOI] [PubMed] [Google Scholar]
  47. Wilson S. P., Kirshner N. Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells. J Biol Chem. 1983 Apr 25;258(8):4994–5000. [PubMed] [Google Scholar]
  48. Wise B. C., Glass D. B., Chou C. H., Raynor R. L., Katoh N., Schatzman R. C., Turner R. S., Kibler R. F., Kuo J. F. Phospholipid-sensitive Ca2+-dependent protein kinase from heart. II. Substrate specificity and inhibition by various agents. J Biol Chem. 1982 Jul 25;257(14):8489–8495. [PubMed] [Google Scholar]
  49. Wojcikiewicz R. J., Nahorski S. R. Phosphoinositide hydrolysis in permeabilized SH-SY5Y human neuroblastoma cells is inhibited by mastoparan. FEBS Lett. 1989 Apr 24;247(2):341–344. doi: 10.1016/0014-5793(89)81366-3. [DOI] [PubMed] [Google Scholar]
  50. Yingst D. R. Modulation of the Na,K-ATPase by Ca and intracellular proteins. Annu Rev Physiol. 1988;50:291–303. doi: 10.1146/annurev.ph.50.030188.001451. [DOI] [PubMed] [Google Scholar]
  51. Yoshimura N., Kikuchi T., Sasaki T., Kitahara A., Hatanaka M., Murachi T. Two distinct Ca2+ proteases (calpain I and calpain II) purified concurrently by the same method from rat kidney. J Biol Chem. 1983 Jul 25;258(14):8883–8889. [PubMed] [Google Scholar]
  52. van Adelsberg J., Al-Awqati Q. Regulation of cell pH by Ca+2-mediated exocytotic insertion of H+-ATPases. J Cell Biol. 1986 May;102(5):1638–1645. doi: 10.1083/jcb.102.5.1638. [DOI] [PMC free article] [PubMed] [Google Scholar]

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