Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Oct 15;319(Pt 2):649–656. doi: 10.1042/bj3190649

Bradykinin B2 receptor-induced and inositol tetrakisphosphate-evoked Ca2+ entry is sensitive to a protein tyrosine phosphorylation inhibitor in ras-transformed NIH/3T3 fibroblasts.

M Hashii 1, S Nakashima 1, S Yokoyama 1, K Enomoto 1, Y Minabe 1, Y Nozawa 1, H Higashida 1
PMCID: PMC1217816  PMID: 8912707

Abstract

Signal transduction from mouse bradykinin B2 receptors to calcium influx was studied in ras-transformed NIH/3T3 (DT) fibroblasts. DT cells were preloaded with fura-2 and whole-cell voltage-clamped. Activation of B2 receptors resulted in a decrease of cellular fluorescence at the excitation wavelength of 340, or 360 nm after MnCl2 application, in both the presence and absence of external Ca2+ in DT cells, at a holding potential of -40 mV. This Mn2+ entry through the Ca2+ influx pathway increased with membrane hyperpolarization. Internal application of inositol 1,3,4,5-tetrakisphosphate (InsP4), but not of inositol 1,4,5-trisphosphate, mimicked membrane potential-dependent Mn2+ entry. Bradykinin- and InsP4-induced Ca2+ influx was blocked by 10-100 microM genistein, a tyrosine kinase inhibitor. B2 receptor activation induced time-dependent tyrosine phosphorylation of mitogen-activated protein kinase and 120 kDa protein, which was dose-dependently inhibited by genistein. Bradykinin was unable to induce Ca2+ oscillations in genistein-treated DT cells. Our results show that bradykinin-induced Ca2+ influx and oscillations depend upon protein tyrosine phosphorylation. The results suggest that two bradykinin B2 receptor-activated signal pathways, protein tyrosine phosphorylation and formation of InsP4, merge at the Ca2+ influx process in ras-transformed NIH/3T3 fibroblasts.

Full Text

The Full Text of this article is available as a PDF (796.3 KB).

Selected References

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

  1. Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanabe S., Itoh N., Shibuya M., Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem. 1987 Apr 25;262(12):5592–5595. [PubMed] [Google Scholar]
  2. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  3. Cheek T. R., Murawsky M. M., Stauderman K. A. Histamine-induced Ca2+ entry precedes Ca2+ mobilization in bovine adrenal chromaffin cells. Biochem J. 1994 Dec 1;304(Pt 2):469–476. doi: 10.1042/bj3040469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Clapham D. E. Calcium signaling. Cell. 1995 Jan 27;80(2):259–268. doi: 10.1016/0092-8674(95)90408-5. [DOI] [PubMed] [Google Scholar]
  6. Cullen P. J., Hsuan J. J., Truong O., Letcher A. J., Jackson T. R., Dawson A. P., Irvine R. F. Identification of a specific Ins(1,3,4,5)P4-binding protein as a member of the GAP1 family. Nature. 1995 Aug 10;376(6540):527–530. doi: 10.1038/376527a0. [DOI] [PubMed] [Google Scholar]
  7. Demaurex N., Monod A., Lew D. P., Krause K. H. Characterization of receptor-mediated and store-regulated Ca2+ influx in human neutrophils. Biochem J. 1994 Feb 1;297(Pt 3):595–601. doi: 10.1042/bj2970595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. Fleming I., Fisslthaler B., Busse R. Calcium signaling in endothelial cells involves activation of tyrosine kinases and leads to activation of mitogen-activated protein kinases. Circ Res. 1995 Apr;76(4):522–529. doi: 10.1161/01.res.76.4.522. [DOI] [PubMed] [Google Scholar]
  10. Fukuda M., Aruga J., Niinobe M., Aimoto S., Mikoshiba K. Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II. J Biol Chem. 1994 Nov 18;269(46):29206–29211. [PubMed] [Google Scholar]
  11. Gawler D. J., Zhang L. J., Moran M. F. Mutation-deletion analysis of a Ca(2+)-dependent phospholipid binding (CaLB) domain within p120 GAP, a GTPase-activating protein for p21 ras. Biochem J. 1995 Apr 15;307(Pt 2):487–491. doi: 10.1042/bj3070487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  13. Hashii M., Hirata M., Ozaki S., Nozawa Y., Higashida H. Ca2+ influx evoked by inositol-3,4,5,6-tetrakisphosphate in ras-transformed NIH/3T3 fibroblasts. FEBS Lett. 1994 Mar 7;340(3):276–280. doi: 10.1016/0014-5793(94)80153-3. [DOI] [PubMed] [Google Scholar]
  14. Hashii M., Hirata M., Ozaki S., Nozawa Y., Higashida H. Ca2+ influx gated by inositol-3,4,5,6-tetrakisphosphate in NIH/3T3 fibroblasts. Biochem Biophys Res Commun. 1994 May 16;200(3):1300–1306. doi: 10.1006/bbrc.1994.1592. [DOI] [PubMed] [Google Scholar]
  15. Hashii M., Nozawa Y., Higashida H. Bradykinin-induced cytosolic Ca2+ oscillations and inositol tetrakisphosphate-induced Ca2+ influx in voltage-clamped ras-transformed NIH/3T3 fibroblasts. J Biol Chem. 1993 Sep 15;268(26):19403–19410. [PubMed] [Google Scholar]
  16. Higashida H., Hashii M., Fukuda K., Caulfield M. P., Numa S., Brown D. A. Selective coupling of different muscarinic acetylcholine receptors to neuronal calcium currents in DNA-transfected cells. Proc Biol Sci. 1990 Oct 22;242(1303):68–74. doi: 10.1098/rspb.1990.0105. [DOI] [PubMed] [Google Scholar]
  17. Hoth M., Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature. 1992 Jan 23;355(6358):353–356. doi: 10.1038/355353a0. [DOI] [PubMed] [Google Scholar]
  18. Huang S., Maher V. M., McCormick J. J. Extracellular Ca2+ stimulates the activation of mitogen-activated protein kinase and cell growth in human fibroblasts. Biochem J. 1995 Sep 15;310(Pt 3):881–885. doi: 10.1042/bj3100881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huckle W. R., Dy R. C., Earp H. S. Calcium-dependent increase in tyrosine kinase activity stimulated by angiotensin II. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8837–8841. doi: 10.1073/pnas.89.18.8837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Inglese J., Koch W. J., Touhara K., Lefkowitz R. J. G beta gamma interactions with PH domains and Ras-MAPK signaling pathways. Trends Biochem Sci. 1995 Apr;20(4):151–156. doi: 10.1016/s0968-0004(00)88992-6. [DOI] [PubMed] [Google Scholar]
  21. Ito A., Satoh T., Kaziro Y., Itoh H. G protein beta gamma subunit activates Ras, Raf, and MAP kinase in HEK 293 cells. FEBS Lett. 1995 Jul 10;368(1):183–187. doi: 10.1016/0014-5793(95)00643-n. [DOI] [PubMed] [Google Scholar]
  22. Jacob R. Agonist-stimulated divalent cation entry into single cultured human umbilical vein endothelial cells. J Physiol. 1990 Feb;421:55–77. doi: 10.1113/jphysiol.1990.sp017933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jenner S., Farndale R. W., Sage S. O. The effect of calcium-store depletion and refilling with various bivalent cations on tyrosine phosphorylation and Mn2+ entry in fura-2-loaded human platelets. Biochem J. 1994 Oct 15;303(Pt 2):337–339. doi: 10.1042/bj3030337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jones S., Brown D. A., Milligan G., Willer E., Buckley N. J., Caulfield M. P. Bradykinin excites rat sympathetic neurons by inhibition of M current through a mechanism involving B2 receptors and G alpha q/11. Neuron. 1995 Feb;14(2):399–405. doi: 10.1016/0896-6273(95)90295-3. [DOI] [PubMed] [Google Scholar]
  25. Kass G. E., Webb D. L., Chow S. C., Llopis J., Berggren P. O. Receptor-mediated Mn2+ influx in rat hepatocytes: comparison of cells loaded with Fura-2 ester and cells microinjected with Fura-2 salt. Biochem J. 1994 Aug 15;302(Pt 1):5–9. doi: 10.1042/bj3020005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kwan C. Y., Putney J. W., Jr Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and Ba2+ of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool. J Biol Chem. 1990 Jan 15;265(2):678–684. [PubMed] [Google Scholar]
  27. Leeb-Lundberg L. M., Song X. H. Bradykinin and bombesin rapidly stimulate tyrosine phosphorylation of a 120-kDa group of proteins in Swiss 3T3 cells. J Biol Chem. 1991 Apr 25;266(12):7746–7749. [PubMed] [Google Scholar]
  28. Leeb-Lundberg L. M., Song X. H., Mathis S. A. Focal adhesion-associated proteins p125FAK and paxillin are substrates for bradykinin-stimulated tyrosine phosphorylation in Swiss 3T3 cells. J Biol Chem. 1994 Sep 30;269(39):24328–24334. [PubMed] [Google Scholar]
  29. Lev S., Moreno H., Martinez R., Canoll P., Peles E., Musacchio J. M., Plowman G. D., Rudy B., Schlessinger J. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature. 1995 Aug 31;376(6543):737–745. doi: 10.1038/376737a0. [DOI] [PubMed] [Google Scholar]
  30. Llopis J., Kass G. E., Gahm A., Orrenius S. Evidence for two pathways of receptor-mediated Ca2+ entry in hepatocytes. Biochem J. 1992 May 15;284(Pt 1):243–247. doi: 10.1042/bj2840243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lückhoff A., Clapham D. E. Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca(2+)-permeable channel. Nature. 1992 Jan 23;355(6358):356–358. doi: 10.1038/355356a0. [DOI] [PubMed] [Google Scholar]
  32. Maekawa M., Li S., Iwamatsu A., Morishita T., Yokota K., Imai Y., Kohsaka S., Nakamura S., Hattori S. A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions. Mol Cell Biol. 1994 Oct;14(10):6879–6885. doi: 10.1128/mcb.14.10.6879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moolenaar W. H. Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem. 1995 Jun 2;270(22):12949–12952. doi: 10.1074/jbc.270.22.12949. [DOI] [PubMed] [Google Scholar]
  34. Morris A. P., Gallacher D. V., Irvine R. F., Petersen O. H. Synergism of inositol trisphosphate and tetrakisphosphate in activating Ca2+-dependent K+ channels. Nature. 1987 Dec 17;330(6149):653–655. doi: 10.1038/330653a0. [DOI] [PubMed] [Google Scholar]
  35. Mozhayeva G. N., Naumov A. P., Kuryshev YuA Variety of Ca(2+)-permeable channels in human carcinoma A431 cells. J Membr Biol. 1991 Nov;124(2):113–126. doi: 10.1007/BF01870456. [DOI] [PubMed] [Google Scholar]
  36. Niinobe M., Yamaguchi Y., Fukuda M., Mikoshiba K. Synaptotagmin is an inositol polyphosphate binding protein: isolation and characterization as an Ins 1,3,4,5-P4 binding protein. Biochem Biophys Res Commun. 1994 Dec 15;205(2):1036–1042. doi: 10.1006/bbrc.1994.2770. [DOI] [PubMed] [Google Scholar]
  37. Noda M., Selinger Z., Scolnick E. M., Bassin R. H. Flat revertants isolated from Kirsten sarcoma virus-transformed cells are resistant to the action of specific oncogenes. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5602–5606. doi: 10.1073/pnas.80.18.5602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sargeant P., Farndale R. W., Sage S. O. ADP- and thapsigargin-evoked Ca2+ entry and protein-tyrosine phosphorylation are inhibited by the tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate in fura-2-loaded human platelets. J Biol Chem. 1993 Aug 25;268(24):18151–18156. [PubMed] [Google Scholar]
  39. Schlaepfer D. D., Hanks S. K., Hunter T., van der Geer P. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature. 1994 Dec 22;372(6508):786–791. doi: 10.1038/372786a0. [DOI] [PubMed] [Google Scholar]
  40. Semenchuk L. A., Di Salvo J. Receptor-activated increases in intracellular calcium and protein tyrosine phosphorylation in vascular smooth muscle cells. FEBS Lett. 1995 Aug 14;370(1-2):127–130. doi: 10.1016/0014-5793(95)00808-m. [DOI] [PubMed] [Google Scholar]
  41. Tepel M., Kühnapfel S., Theilmeier G., Teupe C., Schlotmann R., Zidek W. Filling state of intracellular Ca2+ pools triggers trans plasma membrane Na+ and Ca2+ influx by a tyrosine kinase-dependent pathway. J Biol Chem. 1994 Oct 21;269(42):26239–26242. [PubMed] [Google Scholar]
  42. Tsubokawa H., Oguro K., Robinson H. P., Masuzawa T., Rhee T. S., Takenawa T., Kawai N. Inositol 1,3,4,5-tetrakisphosphate as a mediator of neuronal death in ischemic hippocampus. Neuroscience. 1994 Mar;59(2):291–297. doi: 10.1016/0306-4522(94)90597-5. [DOI] [PubMed] [Google Scholar]
  43. Vostal J. G., Jackson W. L., Shulman N. R. Cytosolic and stored calcium antagonistically control tyrosine phosphorylation of specific platelet proteins. J Biol Chem. 1991 Sep 5;266(25):16911–16916. [PubMed] [Google Scholar]
  44. Wilk-Blaszczak M. A., Singer W. D., Gutowski S., Sternweis P. C., Belardetti F. The G protein G13 mediates inhibition of voltage-dependent calcium current by bradykinin. Neuron. 1994 Nov;13(5):1215–1224. doi: 10.1016/0896-6273(94)90059-0. [DOI] [PubMed] [Google Scholar]
  45. Wolsing D. H., Rosenbaum J. S. The mechanism for the rapid desensitization in bradykinin-stimulated inositol monophosphate production in NG108-15 cells involves interaction of a single receptor with multiple signaling pathways. J Pharmacol Exp Ther. 1993 Jul;266(1):253–261. [PubMed] [Google Scholar]
  46. Yokoyama S., Kimura Y., Taketo M., Black J. A., Ransom B. R., Higashida H. B2 bradykinin receptors in NG108-15 cells: cDNA cloning and functional expression. Biochem Biophys Res Commun. 1994 Apr 15;200(1):634–641. doi: 10.1006/bbrc.1994.1495. [DOI] [PubMed] [Google Scholar]
  47. Zachary I., Sinnett-Smith J., Rozengurt E. Bombesin, vasopressin, and endothelin stimulation of tyrosine phosphorylation in Swiss 3T3 cells. Identification of a novel tyrosine kinase as a major substrate. J Biol Chem. 1992 Sep 25;267(27):19031–19034. [PubMed] [Google Scholar]
  48. van Biesen T., Hawes B. E., Luttrell D. K., Krueger K. M., Touhara K., Porfiri E., Sakaue M., Luttrell L. M., Lefkowitz R. J. Receptor-tyrosine-kinase- and G beta gamma-mediated MAP kinase activation by a common signalling pathway. Nature. 1995 Aug 31;376(6543):781–784. doi: 10.1038/376781a0. [DOI] [PubMed] [Google Scholar]
  49. van Corven E. J., Hordijk P. L., Medema R. H., Bos J. L., Moolenaar W. H. Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1257–1261. doi: 10.1073/pnas.90.4.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES