Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1984 Nov 1;223(3):893–900. doi: 10.1042/bj2230893

Intracellular Ca2+ and phorbol esters synergistically inhibit internalization of epidermal growth factor in pancreatic acini.

C D Logsdon, J A Williams
PMCID: PMC1144377  PMID: 6095811

Abstract

The association of 125I-labelled epidermal growth factor (125I-EGF) with mouse pancreatic acinar cells was inhibited by secretagogues which increase intracellular free Ca2+ concentrations. These agents included cholecystokinin-octapeptide (CCK8) and the Ca2+ ionophore A23187. Inhibition by CCK8 was blocked by lowering the incubation temperature from 37 degrees C to 15 degrees C. Moreover, in contrast with studies of intact acini, the binding of 125I-EGF to isolated acinar membrane particles was not affected either by CCK8, or by varying the level of Ca2+ in the incubation medium. These results indicated, therefore, that the inhibition of 125I-EGF association with acinar cells required intact cells that are metabolically active. Since intact cells at 37 degrees C are known to internalize bound EGF rapidly, acid washing was used to distinguish membrane-associated hormone from internalized hormone. Under steady-state conditions 86% of the 125I-EGF associated with the acini was found to be internalized by this technique. When agents that increased intracellular Ca2+ were tested they all markedly reduced the amount of internalized hormone, whereas surface binding was only minimally affected. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), which is known to activate protein kinase C, a Ca2+-regulated enzyme, also inhibited the association of EGF with acini. This inhibition was similar to that induced by elevated intracellular Ca2+. To test whether these two inhibitory phenomena were related, the effects of TPA in combination with the Ca2+ ionophore A23187 were examined. At low concentrations the effects were synergistic, whereas at high concentrations the maximal level of inhibition was not changed. We suggest therefore that elevated intracellular Ca2+ and phorbol esters may inhibit EGF internalization by a mechanism involving activation of protein kinase C.

Full text

PDF
897

Selected References

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

  1. Brown K. D., Dicker P., Rozengurt E. Inhibition of epidermal growth factor binding to surface receptors by tumor promotors. Biochem Biophys Res Commun. 1979 Feb 28;86(4):1037–1043. doi: 10.1016/0006-291x(79)90221-3. [DOI] [PubMed] [Google Scholar]
  2. Burnham D. B., Williams J. A. Activation of protein kinase activity in pancreatic acini by calcium and cAMP. Am J Physiol. 1984 May;246(5 Pt 1):G500–G508. doi: 10.1152/ajpgi.1984.246.5.G500. [DOI] [PubMed] [Google Scholar]
  3. Carpenter G., Cohen S. Epidermal growth factor. Annu Rev Biochem. 1979;48:193–216. doi: 10.1146/annurev.bi.48.070179.001205. [DOI] [PubMed] [Google Scholar]
  4. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
  5. Cochet C., Gill G. N., Meisenhelder J., Cooper J. A., Hunter T. C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Feb 25;259(4):2553–2558. [PubMed] [Google Scholar]
  6. De Larco J. E., Todaro G. J. Sarcoma growth factor (SGF): specific binding to epidermal growth factor (EGF) membrane receptors. J Cell Physiol. 1980 Feb;102(2):267–277. doi: 10.1002/jcp.1041020218. [DOI] [PubMed] [Google Scholar]
  7. Dembiński A., Gregory H., Konturek S. J., Polański M. Trophic action of epidermal growth factor on the pancreas and gastroduodenal mucosa in rats. J Physiol. 1982 Apr;325:35–42. doi: 10.1113/jphysiol.1982.sp014133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fox C. F., Das M. Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: the endocytic activation hypothesis. J Supramol Struct. 1979;10(2):199–214. doi: 10.1002/jss.400100210. [DOI] [PubMed] [Google Scholar]
  9. Froscio M., Guy G. R., Murray A. W. Calmodulin inhibitors modify cell surface changes triggered by a tumor promoter. Biochem Biophys Res Commun. 1981 Feb 12;98(3):829–835. doi: 10.1016/0006-291x(81)91186-4. [DOI] [PubMed] [Google Scholar]
  10. Goldfine I. D., Smith G. J., Wong K. Y., Jones A. L. Cellular uptake and nuclear binding of insulin in human cultured lymphocytes: evidence for potential intracellular sites of insulin action. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1368–1372. doi: 10.1073/pnas.74.4.1368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gunther G. R. Effect of 12-O-tetradecanoyl-phorbol-13-acetate on Ca2+ efflux and protein discharge in pancreatic acini. J Biol Chem. 1981 Dec 10;256(23):12040–12045. [PubMed] [Google Scholar]
  12. Haigler H. T., Maxfield F. R., Willingham M. C., Pastan I. Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. J Biol Chem. 1980 Feb 25;255(4):1239–1241. [PubMed] [Google Scholar]
  13. Hems D. A., Whitton P. D. Control of hepatic glycogenolysis. Physiol Rev. 1980 Jan;60(1):1–50. doi: 10.1152/physrev.1980.60.1.1. [DOI] [PubMed] [Google Scholar]
  14. Iwashita S., Fox C. F. Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells. J Biol Chem. 1984 Feb 25;259(4):2559–2567. [PubMed] [Google Scholar]
  15. Johnson L. R. Effects of gastrointestinal hormones on pancreatic growth. Cancer. 1981 Mar 15;47(6 Suppl):1640–1645. doi: 10.1002/1097-0142(19810315)47:6+<1640::aid-cncr2820471430>3.0.co;2-s. [DOI] [PubMed] [Google Scholar]
  16. King A. C., Cuatrecasas P. Peptide hormone-induced receptor mobility, aggregation, and internalization. N Engl J Med. 1981 Jul 9;305(2):77–88. doi: 10.1056/NEJM198107093050206. [DOI] [PubMed] [Google Scholar]
  17. Korc M., Matrisian L. M., Magun B. E. Cytosolic calcium regulates epidermal growth factor endocytosis in rat pancreas and cultured fibroblasts. Proc Natl Acad Sci U S A. 1984 Jan;81(2):461–465. doi: 10.1073/pnas.81.2.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Korc M., Matrisian L. M., Magun B. E. Direct modulation of epidermal growth factor binding by cholecystokinin. Life Sci. 1983 Aug 8;33(6):561–568. doi: 10.1016/0024-3205(83)90131-5. [DOI] [PubMed] [Google Scholar]
  19. Korc M., Matrisian L. M., Planck S. R., Magun B. E. Binding of epidermal growth factor in rat pancreatic acini. Biochem Biophys Res Commun. 1983 Mar 29;111(3):1066–1073. doi: 10.1016/0006-291x(83)91408-0. [DOI] [PubMed] [Google Scholar]
  20. Lee L. S., Weinstein I. B. Mechanism of tumor promoter inhibition of cellular binding of epidermal growth factor. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5168–5172. doi: 10.1073/pnas.76.10.5168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lee L. S., Weinstein I. B. Tumor-promoting phorbol esters inhibit binding of epidermal growth factor to cellular receptors. Science. 1978 Oct 20;202(4365):313–315. doi: 10.1126/science.308698. [DOI] [PubMed] [Google Scholar]
  22. Logsdon C. D., Williams J. A. Epidermal growth factor binding and biologic effects on mouse pancreatic acini. Gastroenterology. 1983 Aug;85(2):339–345. [PubMed] [Google Scholar]
  23. Logsdon C. D., Williams J. A. Epidermal growth factor: intracellular Ca2+ inhibits its association with pancreatic acini and A431 cells. FEBS Lett. 1983 Dec 12;164(2):335–339. doi: 10.1016/0014-5793(83)80312-3. [DOI] [PubMed] [Google Scholar]
  24. Logsdon C. D., Williams J. A. Pancreatic acini in short-term culture: regulation by EGF, carbachol, insulin, and corticosterone. Am J Physiol. 1983 Jun;244(6):G675–G682. doi: 10.1152/ajpgi.1983.244.6.G675. [DOI] [PubMed] [Google Scholar]
  25. Magun B. E., Matrisian L. M., Bowden G. T. Epidermal growth factor. Ability of tumor promoter to alter its degradation, receptor affinity and receptor number. J Biol Chem. 1980 Jul 10;255(13):6373–6381. [PubMed] [Google Scholar]
  26. Murray A. W., Froscio M. Inhibition of epidermal growth factor binding to cultured mouse epidermal cells by tumor promoters. Carcinogenesis. 1980 Aug;1(8):681–684. doi: 10.1093/carcin/1.8.681. [DOI] [PubMed] [Google Scholar]
  27. Naka M., Nishikawa M., Adelstein R. S., Hidaka H. Phorbol ester-induced activation of human platelets is associated with protein kinase C phosphorylation of myosin light chains. Nature. 1983 Dec 1;306(5942):490–492. doi: 10.1038/306490a0. [DOI] [PubMed] [Google Scholar]
  28. Niedel J. E., Kuhn L. J., Vandenbark G. R. Phorbol diester receptor copurifies with protein kinase C. Proc Natl Acad Sci U S A. 1983 Jan;80(1):36–40. doi: 10.1073/pnas.80.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ochs D. L., Korenbrot J. I., Williams J. A. Intracellular free calcium concentrations in isolated pancreatic acini; effects of secretagogues. Biochem Biophys Res Commun. 1983 Nov 30;117(1):122–128. doi: 10.1016/0006-291x(83)91549-8. [DOI] [PubMed] [Google Scholar]
  30. Ozanne B., Fulton R. J., Kaplan P. L. Kirsten murine sarcoma virus transformed cell lines and a spontaneously transformed rat cell-line produce transforming factors. J Cell Physiol. 1980 Oct;105(1):163–180. doi: 10.1002/jcp.1041050118. [DOI] [PubMed] [Google Scholar]
  31. Pfeffer S. R., Drubin D. G., Kelly R. B. Identification of three coated vesicle components as alpha- and beta-tubulin linked to a phosphorylated 50,000-dalton polypeptide. J Cell Biol. 1983 Jul;97(1):40–47. doi: 10.1083/jcb.97.1.40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rink T. J., Sanchez A., Hallam T. J. Diacylglycerol and phorbol ester stimulate secretion without raising cytoplasmic free calcium in human platelets. Nature. 1983 Sep 22;305(5932):317–319. doi: 10.1038/305317a0. [DOI] [PubMed] [Google Scholar]
  33. Rozengurt E., Brown K. D., Pettican P. Vasopressin inhibition of epidermal growth factor binding to cultured mouse cells. J Biol Chem. 1981 Jan 25;256(2):716–722. [PubMed] [Google Scholar]
  34. Rozengurt E., Collins M., Brown K. D., Pettican P. Inhibition of epidermal growth factor binding to mouse cultured cells by fibroblast-derived growth factor. Evidence for an indirect mechanism. J Biol Chem. 1982 Apr 10;257(7):3680–3686. [PubMed] [Google Scholar]
  35. Salomon D. S. Inhibition of epidermal growth factor binding to mouse embryonal carcinoma cells by phorbol esters mediated by specific phorbol ester receptors. J Biol Chem. 1981 Aug 10;256(15):7958–7966. [PubMed] [Google Scholar]
  36. Savion N., Vlodavsky I., Gospodarowicz D. Role of the degradation process in the mitogenic effect of epidermal growth factor. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1466–1470. doi: 10.1073/pnas.77.3.1466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shoyab M., De Larco J. E., Todaro G. J. Biologically active phorbol esters specifically alter affinity of epidermal growth factor membrane receptors. Nature. 1979 May 31;279(5712):387–391. doi: 10.1038/279387a0. [DOI] [PubMed] [Google Scholar]
  38. Steigerwalt R. W., Williams J. A. Characterization of cholecystokinin receptors on rat pancreatic membranes. Endocrinology. 1981 Nov;109(5):1746–1753. doi: 10.1210/endo-109-5-1746. [DOI] [PubMed] [Google Scholar]
  39. Williams J. A. Regulation of pancreatic acinar cell function by intracellular calcium. Am J Physiol. 1980 Apr;238(4):G269–G279. doi: 10.1152/ajpgi.1980.238.4.G269. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES