Advertisement

Pflügers Archiv

, Volume 429, Issue 6, pp 805–808 | Cite as

Cholecystokinin-octapeptide affects the fluorescence signal of a single pancreatic acinar cell loaded with the acrylodan-labelled MARCKS peptide, a protein kinase C substrate

  • A. Ngezahayo
  • F. Lang
  • H. -A. Kolb
Original Article Transport Processes, Metabolism and Endocrinology; Kidney, Gastrointestinal Tract, and Exocrine Glands

Abstract

We used a fluorescent derivative of the myristoylated, alanine-rich C kinase substrate (MARCKS) peptide as a probe for protein kinase C (PKC) activation by cholecystokinin-octapeptide (CCK-8) in isolated pancreatic acinar cell pairs. The diffusion of the acrylodan-labelled MARCKS-peptide into the cell interior could be monitored by the increase of fluorescence in the whole-cell patch-clamp configuration. Addition of 10 pM CCK-8 to the bath induced repetitive fluctuations of the fluorescent signal in the time range of 4–5 min. With 1 nM CCK-8 a sustained decrease of the signal was observed. Addition of polymyxin B, a specific inhibitor of PKC activation, to the pipette filling solution suppressed the CCK-8-induced change of fluorescence. The data indicate activation of PKC by CCK-8 in pancreatic acinar cells and could be compared with the previously studied CCK-8-induced gap junction uncoupling.

Key words

Fluorescent MARCKS peptide Protein kinase C activation Cholecystokinin Pancreatic acinar cells Patch-clamp 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Asaoka Y, Yoshida K, Oka M, Shinomura T, Koide H, Ogita K, Kikkawa U, Nishizuka Y (1992) The family of protein kinase C in transmembrane signalling for cellular regulation. J Nutr Sci Vitaminol (Tokyo) (Spec No):7–12Google Scholar
  2. 2.
    Chanson M, Meda P, Bruzzone R (1989) Increase in pancreatic secretion during uncoupling: evidence for a protein kinase C-independent effect. Exptl Cell Res 182:349–357Google Scholar
  3. 3.
    Graff JM, Rajan RR, Randall RR, Nairn AC, Blackshear PJ (1991) Protein kinase C substrate and inhibitor characteristics of peptides from the myristoylated alanine-rich C kinase substrate (MARCKS) protein phosphorylation site domain. J Biol Chem 266:14390–14398Google Scholar
  4. 4.
    Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp technique for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100Google Scholar
  5. 5.
    Hartwig JH, Thelen M, Rosen A, Janmey PA, Nairn AC, Aderem A (1992) MARCKS is an actin filament cross linking protein regulated by protein kinase C and calcium-calmodulin. Nature 356:618–622Google Scholar
  6. 6.
    Jensen RT, Wank SA, Rowley WH, Sato S, Gardner JD (1989) Interaction of CCK with pancreatic acinar cells. Trends Pharmacol Sci 10:418–423Google Scholar
  7. 7.
    Ngezahayo A, Kolb HA (1993) Gap junctional conductance tunes phase difference of cholecystokinin evoked calcium oscillations in pairs of pancreatic acinar cells. Pflügers Arch 422:413–415Google Scholar
  8. 8.
    Ngezahayo A, Kolb HA (1994) Regulation of gap junctional coupling in isolated pancreatic acinar cell pairs by cholecystokinin-octapeptide, vasoactive intestinal peptide (VIP) and a VIP-antagonist, J Membr Biol 139:127–136Google Scholar
  9. 9.
    McIlroy BK, Walters JD, Johnson JD (1991) A continuous fluorescence assay for protein kinase C. Anal Biochem 195:148–152Google Scholar
  10. 10.
    McIlroy BK, Walters JD, Blackshear PJ, Johnson JD (1991) Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin. J Biol Chem 266:4959–4964Google Scholar
  11. 11.
    Somogyi R, Batzer A, Kolb HA (1989) Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C. J Membr Biol 108:273–282Google Scholar
  12. 12.
    Toescu EC, Lawrie AM, Petersen OH, Gallacher DV (1992) Spatial and temporal distribution of agonist-evoked cytoplasmic Ca2+ signals in exocrine cells analysed by digital image microscopy. EMBO J 11:1623–1629Google Scholar
  13. 13.
    Walsh JW (1987) Gastrointestinal hormones. In: Johnson LR (ed) Physiology of the gastrointestinal track, 2nd edn. Raven Press, New York, pp 181–253Google Scholar
  14. 14.
    Wrenn RW, Wooten MW (1984) Dual calcium-dependent protein phosphorylation systems in pancreas and their differential regulation of polymyxin B1. Life Sci 35:267–276Google Scholar
  15. 15.
    Wu WS, Walaas SI, Nairn AC, Greengard P (1982) Calcium/phospholipid regulates phosphorylation of a Mr“87k” substrate protein in brain synaptosomes. Proc Natl Acad Sci USA 79:5249–5253Google Scholar
  16. 16.
    Yule DI, Williams JA (1992) U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells. J Biol Chem 267:13830–13835Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • A. Ngezahayo
    • 1
  • F. Lang
    • 1
  • H. -A. Kolb
    • 1
  1. 1.University of TübingenInstitute of Physiology ITübingenGermany

Personalised recommendations