Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 May 1;110(5):1803–1811. doi: 10.1083/jcb.110.5.1803

Control of intracellular pH and growth by fibronectin in capillary endothelial cells

PMCID: PMC2200182  PMID: 2159481

Abstract

The aim of this work was to analyze the mechanism by which fibronectin (FN) regulates capillary endothelial cell proliferation. Endothelial cell growth can be controlled in chemically-defined medium by varying the density of FN coated on the substratum (Ingber, D. E., and J. Folkman. J. Cell Biol. 1989. 109:317-330). In this system, DNA synthetic rates are stimulated by FN in direct proportion to its effect on cell extension (projected cell areas) both in the presence and absence of saturating amounts of basic FGF. To investigate direct growth signaling by FN, we carried out microfluorometric measurements of intracellular pH (pHi), a cytoplasmic signal that is commonly influenced by soluble mitogens. pHi increased 0.18 pH units as FN coating densities were raised and cells progressed from round to spread. Intracellular alkalinization induced by attachment to FN was rapid and followed the time course of cell spreading. When measured in the presence and absence of FGF, the effects of FN and FGF on pHi were found to be independent and additive. Furthermore, DNA synthesis correlated with pHi for all combinations of FGF and FN. Ethylisopropylamiloride, a specific inhibitor of the plasma membrane Na+/H+ antiporter, completely suppressed the effects of FN on both pHi and DNA synthesis. However, cytoplasmic pH per se did not appear to be a critical determinant of growth since DNA synthesis was not significantly inhibited when pHi was lowered over the physiological range by varying the pH of the medium. We conclude that FN and FGF exert their growth-modulating effects in part through activation of the Na+/H+ exchanger, although they appear to trigger this system via separate pathways.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Ausprunk D. H., Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res. 1977 Jul;14(1):53–65. doi: 10.1016/0026-2862(77)90141-8. [DOI] [PubMed] [Google Scholar]
  2. Banga H. S., Simons E. R., Brass L. F., Rittenhouse S. E. Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9197–9201. doi: 10.1073/pnas.83.23.9197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bierman A. J., Cragoe E. J., Jr, de Laat S. W., Moolenaar W. H. Bicarbonate determines cytoplasmic pH and suppresses mitogen-induced alkalinization in fibroblastic cells. J Biol Chem. 1988 Oct 25;263(30):15253–15256. [PubMed] [Google Scholar]
  4. Boyarsky G., Ganz M. B., Sterzel R. B., Boron W. F. pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3-. Am J Physiol. 1988 Dec;255(6 Pt 1):C844–C856. doi: 10.1152/ajpcell.1988.255.6.C844. [DOI] [PubMed] [Google Scholar]
  5. Burns C. P., Rozengurt E. Extracellular Na+ and initiation of DNA synthesis: role of intracellular pH and K+. J Cell Biol. 1984 Mar;98(3):1082–1089. doi: 10.1083/jcb.98.3.1082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Denhardt D. T., Edwards D. R., Parfett C. L. Gene expression during the mammalian cell cycle. Biochim Biophys Acta. 1986 Oct 28;865(2):83–125. doi: 10.1016/0304-419x(86)90024-7. [DOI] [PubMed] [Google Scholar]
  7. Doppler W., Jaggi R., Groner B. Induction of v-mos and activated Ha-ras oncogene expression in quiescent NIH 3T3 cells causes intracellular alkalinisation and cell-cycle progression. Gene. 1987;54(1):147–153. doi: 10.1016/0378-1119(87)90357-x. [DOI] [PubMed] [Google Scholar]
  8. Dynan W. S. Modularity in promoters and enhancers. Cell. 1989 Jul 14;58(1):1–4. doi: 10.1016/0092-8674(89)90393-0. [DOI] [PubMed] [Google Scholar]
  9. Esch F., Baird A., Ling N., Ueno N., Hill F., Denoroy L., Klepper R., Gospodarowicz D., Böhlen P., Guillemin R. Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6507–6511. doi: 10.1073/pnas.82.19.6507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Folkman J. Angiogenesis: initiation and control. Ann N Y Acad Sci. 1982;401:212–227. doi: 10.1111/j.1749-6632.1982.tb25720.x. [DOI] [PubMed] [Google Scholar]
  11. Folkman J., Haudenschild C. C., Zetter B. R. Long-term culture of capillary endothelial cells. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5217–5221. doi: 10.1073/pnas.76.10.5217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Folkman J., Moscona A. Role of cell shape in growth control. Nature. 1978 Jun 1;273(5661):345–349. doi: 10.1038/273345a0. [DOI] [PubMed] [Google Scholar]
  13. Gospodarowicz D., Greenburg G., Birdwell C. R. Determination of cellular shape by the extracellular matrix and its correlation with the control of cellular growth. Cancer Res. 1978 Nov;38(11 Pt 2):4155–4171. [PubMed] [Google Scholar]
  14. Grinstein S., Rotin D., Mason M. J. Na+/H+ exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biochim Biophys Acta. 1989 Jan 18;988(1):73–97. doi: 10.1016/0304-4157(89)90004-x. [DOI] [PubMed] [Google Scholar]
  15. Guharay F., Sachs F. Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. J Physiol. 1984 Jul;352:685–701. doi: 10.1113/jphysiol.1984.sp015317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hynes R. O. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. doi: 10.1016/0092-8674(87)90233-9. [DOI] [PubMed] [Google Scholar]
  17. Ingber D. E., Folkman J. Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix. J Cell Biol. 1989 Jul;109(1):317–330. doi: 10.1083/jcb.109.1.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ingber D. E., Madri J. A., Folkman J. A possible mechanism for inhibition of angiogenesis by angiostatic steroids: induction of capillary basement membrane dissolution. Endocrinology. 1986 Oct;119(4):1768–1775. doi: 10.1210/endo-119-4-1768. [DOI] [PubMed] [Google Scholar]
  19. Ingber D. E., Madri J. A., Folkman J. Endothelial growth factors and extracellular matrix regulate DNA synthesis through modulation of cell and nuclear expansion. In Vitro Cell Dev Biol. 1987 May;23(5):387–394. doi: 10.1007/BF02620997. [DOI] [PubMed] [Google Scholar]
  20. Ingber D., Folkman J. Inhibition of angiogenesis through modulation of collagen metabolism. Lab Invest. 1988 Jul;59(1):44–51. [PubMed] [Google Scholar]
  21. L'Allemain G., Franchi A., Cragoe E., Jr, Pouysségur J. Blockade of the Na+/H+ antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series. J Biol Chem. 1984 Apr 10;259(7):4313–4319. [PubMed] [Google Scholar]
  22. Lansman J. B., Hallam T. J., Rink T. J. Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers? 1987 Feb 26-Mar 4Nature. 325(6107):811–813. doi: 10.1038/325811a0. [DOI] [PubMed] [Google Scholar]
  23. Maroudas N. G. Chemical and mechanical requirements for fibroblast adhesion. Nature. 1973 Aug 10;244(5415):353–354. doi: 10.1038/244353a0. [DOI] [PubMed] [Google Scholar]
  24. Maroudas N. G. Growth of fibroblasts on linear and planar anchorages of limiting dimensions. Exp Cell Res. 1973 Sep;81(1):104–110. doi: 10.1016/0014-4827(73)90116-x. [DOI] [PubMed] [Google Scholar]
  25. Moenner M., Magnaldo I., L'Allemain G., Barritault D., Pouysségur J. Early and late mitogenic events induced by FGF on bovine epithelial lens cells are not triggered by hydrolysis of polyphosphoinositides. Biochem Biophys Res Commun. 1987 Jul 15;146(1):32–40. doi: 10.1016/0006-291x(87)90686-3. [DOI] [PubMed] [Google Scholar]
  26. Moolenaar W. H. Effects of growth factors on intracellular pH regulation. Annu Rev Physiol. 1986;48:363–376. doi: 10.1146/annurev.ph.48.030186.002051. [DOI] [PubMed] [Google Scholar]
  27. Musgrove E., Seaman M., Hedley D. Relationship between cytoplasmic pH and proliferation during exponential growth and cellular quiescence. Exp Cell Res. 1987 Sep;172(1):65–75. doi: 10.1016/0014-4827(87)90093-0. [DOI] [PubMed] [Google Scholar]
  28. Ober S. S., Pardee A. B. Both protein kinase C and calcium mediate activation of the Na+/H+ antiporter in Chinese hamster embryo fibroblasts. J Cell Physiol. 1987 Aug;132(2):311–317. doi: 10.1002/jcp.1041320216. [DOI] [PubMed] [Google Scholar]
  29. Panet R., Amir I., Snyder D., Zonenshein L., Atlan H., Laskov R., Panet A. Effect of Na + flux inhibitors on induction of c-fos, c-myc, and ODC genes during cell cycle. J Cell Physiol. 1989 Jul;140(1):161–168. doi: 10.1002/jcp.1041400119. [DOI] [PubMed] [Google Scholar]
  30. Pouysségur J., Sardet C., Franchi A., L'Allemain G., Paris S. A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4833–4837. doi: 10.1073/pnas.81.15.4833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rudland P. S., Jimenez de Asua L. Action of growth factors in the cell cycle. Biochim Biophys Acta. 1979 Feb 4;560(1):91–133. doi: 10.1016/0304-419x(79)90004-0. [DOI] [PubMed] [Google Scholar]
  32. Ruoslahti E., Pierschbacher M. D. New perspectives in cell adhesion: RGD and integrins. Science. 1987 Oct 23;238(4826):491–497. doi: 10.1126/science.2821619. [DOI] [PubMed] [Google Scholar]
  33. Salomon D. S., Liotta L. A., Kidwell W. R. Differential response to growth factor by rat mammary epithelium plated on different collagen substrata in serum-free medium. Proc Natl Acad Sci U S A. 1981 Jan;78(1):382–386. doi: 10.1073/pnas.78.1.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schwartz M. A., Both G., Lechene C. Effect of cell spreading on cytoplasmic pH in normal and transformed fibroblasts. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4525–4529. doi: 10.1073/pnas.86.12.4525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schwartz M. A., Cragoe E. J., Jr, Lechene C. P. pH regulation in spread cells and round cells. J Biol Chem. 1990 Jan 25;265(3):1327–1332. [PubMed] [Google Scholar]
  36. Schwartz M. A., Rupp E. E., Frangioni J. V., Lechene C. P. Cytoplasmic pH and anchorage-independent growth induced by v-Ki-ras, v-src or polyoma middle T. Oncogene. 1990 Jan;5(1):55–58. [PubMed] [Google Scholar]
  37. Schweigerer L., Neufeld G., Friedman J., Abraham J. A., Fiddes J. C., Gospodarowicz D. Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature. 1987 Jan 15;325(6101):257–259. doi: 10.1038/325257a0. [DOI] [PubMed] [Google Scholar]
  38. Shing Y., Folkman J., Haudenschild C., Lund D., Crum R., Klagsbrun M. Angiogenesis is stimulated by a tumor-derived endothelial cell growth factor. J Cell Biochem. 1985;29(4):275–287. doi: 10.1002/jcb.240290402. [DOI] [PubMed] [Google Scholar]
  39. Szwergold B. S., Brown T. R., Freed J. J. Bicarbonate abolishes intracellular alkalinization in mitogen-stimulated 3T3 cells. J Cell Physiol. 1989 Feb;138(2):227–235. doi: 10.1002/jcp.1041380203. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Vicentini L. M., Villereal M. L. Inositol phosphates turnover, cytosolic Ca++ and pH: putative signals for the control of cell growth. Life Sci. 1986 Jun 23;38(25):2269–2276. doi: 10.1016/0024-3205(86)90632-6. [DOI] [PubMed] [Google Scholar]
  42. Vigne P., Frelin C., Cragoe E. J., Jr, Lazdunski M. Ethylisopropyl-amiloride: a new and highly potent derivative of amiloride for the inhibition of the Na+/H+ exchange system in various cell types. Biochem Biophys Res Commun. 1983 Oct 14;116(1):86–90. doi: 10.1016/0006-291x(83)90384-4. [DOI] [PubMed] [Google Scholar]
  43. Werb Z., Tremble P. M., Behrendtsen O., Crowley E., Damsky C. H. Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. J Cell Biol. 1989 Aug;109(2):877–889. doi: 10.1083/jcb.109.2.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Williams R. J. Ion pumps and cell shapes. Trends Biochem Sci. 1988 Jul;13(7):249–249. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES