The Journal of Membrane Biology

, Volume 81, Issue 3, pp 241–253 | Cite as

Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas

  • H. Streb
  • E. Bayerdörffer
  • W. Haase
  • R. F. Irvine
  • I. Schulz


We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983,Nature (London)306:67–69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000×g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of IP3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r=0.96, 1.00, 0.91, respectively) and NADPH cytochromec reductase (r=0.63, 0.98, 090, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochromec oxidase (r=−0.64) and glutamate dehydrogenase (r=−0.75) and with the plasma membrane markers (Na++K+)-ATPase (r=−0.81) and alkaline phosphatase (r=−0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.

Key Words

stimulus secretion coupling pancreatic acinar cells intracellular calcium stores calcium transport inositol phosphates 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Affolter, H., Sigel, E. 1979. A simple system for the measurement of ion activities with solvent polymeric membrane electrodes.Anal. Biochem. 97:315–319PubMedGoogle Scholar
  2. 2.
    Agranoff, B.W., Murphy, P., Seguin, E.B. 1983. Thrombin-induced phosphodiesteratic cleavage of phosphatidylinositol bisphosphate in human platelets.J. Biol. Chem. 258:2076–2078PubMedGoogle Scholar
  3. 3.
    Amsterdam, A., Jamieson, J.D. 1972. Structural and functional characterization of isolated pancreatic exocrine cells.Proc. Natl. Acad. Sci. USA 69:3028–3032PubMedGoogle Scholar
  4. 4.
    Bayerdörffer, E., Streb, H., Eckhardt, L., Haase, W., Schulz, I. 1984. Characterization of calcium uptake into rough endoplasmic reticulum of rat pancreas.J. Membrane Biol. (in press) Google Scholar
  5. 5.
    Berridge, M.J. 1981. Phosphatidylinositol hydrolysis: A multifunctional transducing mechanism.Mol. Cell. Endocrinol. 24:115–140PubMedGoogle Scholar
  6. 6.
    Berridge, M.J. 1983. Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol.Biochem. J. 212:849–858PubMedGoogle Scholar
  7. 7.
    Berridge, M.J., Dawson, M.C., Downes, C.P., Heslop, J.P., Irvine, R.F. 1983. Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.Biochem. J. 212:473–482PubMedGoogle Scholar
  8. 8.
    Billah, M.M., Lapetina, E.G. 1982. Rapid decrease of phosphatidylinostiol 4,5-bisphosphate in thrombin-stimulated platelets.J. Biol. Chem. 257:12705–12708PubMedGoogle Scholar
  9. 9.
    Booth, A.G., Kenny, A.J. 1974. A rapid method for the preparation of microvilli from rabbit kidney.Biochem. J. 142:575–581PubMedGoogle Scholar
  10. 10.
    Creba, J.A., Downes, C.P., Hawkins, P.T., Brewster, G., Michell, R.H., Kirk, C.J. 1983. Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+ mobilizing hormones.Biochem. J. 212:733–747PubMedGoogle Scholar
  11. 11.
    Downes, C.P., Wusteman, M.M. 1983. Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands.Biochem. J. 216:633–640PubMedGoogle Scholar
  12. 12.
    Endo, M. 1977. Calcium release from the sarcoplasmic reticulum.Physiol. Rev. 57:71–108PubMedGoogle Scholar
  13. 13.
    Grado, C., Ballou, C.E. 1961. Myo-inositol phosphates obtained by alkaline hydrolysis of beef brain phosphoinositide.J. Biol. Chem. 236:54–60PubMedGoogle Scholar
  14. 14.
    Hatcher, D.W., Goldstein, G. 1969. Improved methods for determination of RNA and DNA.Anal. Biochem. 31:42–50PubMedGoogle Scholar
  15. 15.
    Joseph, S.K., Thomas, A.P., Williams, R.J., Irvine, R.F., Williamson, J.R. 1984. Myo-inositol 1,4,5-trisphosphate: A second messenger for the hormonal mobilization of intracellular Ca2+ in liver.J. Biol. Chem. 259:3077–3081PubMedGoogle Scholar
  16. 16.
    Kirk, C.J., Creba, J.A., Downes, P., Michell, R.A. 1981. Hormone-stimulated metabolism of inositol lipids and its relationship to hepatic receptor function.Biochem. Soc. Trans. 9:377–379PubMedGoogle Scholar
  17. 17.
    Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193:265–275PubMedGoogle Scholar
  18. 18.
    Martin, T.F.J. 1983. Thyrotropin-releasing hormone rapidly activates the phosphodiester hydrolysis of polyphosphoinositides in GH3 pituitary cells.J. Biol. Chem. 258:14816–14822PubMedGoogle Scholar
  19. 19.
    Michell, R.H. 1975. Inositol phospholipids and cell surface receptor function.Biochim. Biophys. Acta 415:81–147PubMedGoogle Scholar
  20. 20.
    Milutinović, S., Sachs, G., Haase, W., Schulz, I. 1977. Studies on isolated subcellular components of cat pancreas: I. Isolation and enzymatic characterization.J. Membrane Biol. 36:253–279Google Scholar
  21. 21.
    Orchard, J.L., Davis, J.S., Larson, R.E., Farese, R.V. 1984. Effects of carbachol and pancreozymin (cholecystokinin-octapeptide) on polyphosphoinositide metabolism in the rat pancreasin vitro.Biochem. J. 217:281–287PubMedGoogle Scholar
  22. 22.
    Putney, J.W., Burgess, G.M., Halenda, S.P., McKinney, J.S., Rubin, R.P. 1983. Effects of secretagogues on (32P) phosphatidylinositol 4,5-bisphosphate metabolism in the exocrine pancreas.Biochem. J. 212:483–488PubMedGoogle Scholar
  23. 23.
    Rhodes, D., Prpic, V., Exton, J.H., Blackmore, P.F. 1983. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in hepatocytes by vasopressin.J. Biol. Chem. 258:2770–2773PubMedGoogle Scholar
  24. 24.
    Scharschmidt, B.F., Keeffe, E.B., Blankenship, N.M., Ockner, R.K. 1979. Validation of a recording spectrophotometric method for measurement of membrane-associated Mg-and NaK-ATPase activity.J. Lab. Clin. Med. 93:790–799PubMedGoogle Scholar
  25. 25.
    Schmidt, E. 1970. Glutamat-Dehydrogenase UV-Test.In: Methoden der enzymatischen Analyse. H.U. Bergmeyer, editor. pp. 607–613. Verlag Chemie, WeinheimGoogle Scholar
  26. 26.
    Schulz, I. 1980. Messenger role of calcium in function of pancreatic acinar cells.Am. J. Physiol. 239:G335-G347PubMedGoogle Scholar
  27. 27.
    Sottocasa, G.L., Kuylenstierna, B., Ernster, L., Bergstrand, A. 1967. An electron-transport system associated with the outer membrane of liver mitochondria.J. Cell Biol. 32:415–438PubMedGoogle Scholar
  28. 28.
    Streb, H., Irvine, R.F., Berridge, M.J., Schulz, I. 1983. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate.Nature (London) 306:67–69Google Scholar
  29. 29.
    Streb, H., Schulz, I. 1983. Regulation of cytosolic free Ca2+ concentration in acinar cells of rat pancreas.Am. J. Physiol. 245:G347-G357PubMedGoogle Scholar
  30. 30.
    Thomas, A.P., Marks, J.S., Coll, K.E., Williamson, J.R. 1983. Quantitation and early kinetics of inositol lipid changes induced by vasopressin in isolated and cultured hepatocytes.J. Biol. Chem. 258:5716–5725PubMedGoogle Scholar
  31. 31.
    Volpi, M., Yassin, R., Naccache, P.H., Sha'afi, R.I. 1983. Chemotactic factor causes rapid decreases in phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate in rabbit neutrophils.Biochem. Biophys. Res. Commun. 112:957–964PubMedGoogle Scholar
  32. 32.
    Wakasugi, H., Stolze, H., Haase, W., Schulz, I. 1981. Effect of La3+ on secretagogue-induced Ca2+ fluxes in rat isolated pancreatic acinar cells.Am. J. Physiol. 240:G281-G289PubMedGoogle Scholar
  33. 33.
    Weiss, S.J., McKinney, J.S., Putney, J.W. 1982. Receptor-mediated net breakdown of phosphatidylinositol 4,5-bisphosphate in parotid acinar cells.Biochem. J. 206:555–560PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • H. Streb
    • 1
  • E. Bayerdörffer
    • 1
  • W. Haase
    • 1
  • R. F. Irvine
    • 2
  • I. Schulz
    • 1
  1. 1.Max-Planck-Institut für BiophysikFrankfurt (Main) 70Federal Republic of Germany
  2. 2.Department of BiochemistryARC Institute of Animal PhysiologyCambridgeEngland

Personalised recommendations