Skip to main content
SpringerLink
Log in

Characterization of AT2 Receptor Expression in NIH 3T3 Fibroblasts

  • Published:
Cellular and Molecular Neurobiology Aims and scope Submit manuscript

Abstract

1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts.

2. Characterization with selective ligands, dithiothreitol, and GTPγS, indicated that only the AT2 subtype was expressed.

3. AT2 receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT2 receptors being expressed earlier than ACE. In contrast, high expression of AT2 receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras.

4. Our results imply a possible relation of AT2 receptors to cell growth and cell–cell contact.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

REFERENCES

  • Bottari, S. P., de Gasparo, M., Steckelings, U. M., and Levens, N. R. (1993). Angiotensin II receptor subtypes: Characterization, signalling mechanisms, and possible physiological implications. Frontiers Neuroendocrinol. 14:123–171.

    Google Scholar 

  • Bumpus, F. M., Catt, K. J., Chiu, A. T., de Gasparo, M., Goodfriend, T., Husain, A., Peach, M. J., Taylor, D. G., Jr., and Timmermans, P. B. M. W. M. (1991). Nomenclature for angiotensin receptors: A report of the nomenclature committee of the council for high blood pressure research. Hypertension 17:720–723.

    Google Scholar 

  • Chiu, A. T., Duncia, J. V., McCall, D. E., Wong, P. C., Price, W. A., Thoolen, M. J. M. C., Carini, D. J., Johnson, A. L., and Timmermans, P. B. M. W. M. (1989a). Nonpeptide angiotensin II receptor antagonists. III. Structure-function studies. J. Pharmacol. Exp. Ther. 250:867–874.

    Google Scholar 

  • Chiu, A. T., Herblin, W. F., McCall, D. E., Ardecky, R. J., Carini, D. J., Duncia, J. V., Pease, L. J., Wong, P. C., Wexler, R. R., Johnson, A. L., and Timmermans, P. B. M. W. M. (1989b). Identification of angiotensin II receptor subtypes. Biochem. Biophys. Res. Commun. 165:196–203.

    Google Scholar 

  • de Gasparo, M., Husain, A., Alexander, W., Catt, K. J., Chiu, A. T., Drew, M., Goodfriend, T., Harding, J. W., Inagami, T., and Timmermans, P. B. M. W. M. (1995). Proposed update of angiotensin receptor nomenclature. Hypertension 25:924–927.

    Google Scholar 

  • Dudley, D. T., and Summerfelt, R. M. (1993). Regulated expression of angiotensin II (AT2) binding sites in R3T3 cells. Regul Peptides 11:13–28.

    Google Scholar 

  • Dudley, D. T., Hubbell, S. E. E., and Summerfelt, R. M. (1991). Characterization of angiotensin II (AT2) binding sites in R3T3 cells. Mol. Pharmacol. 40:360–367.

    Google Scholar 

  • Dzau, V. J., Sasamura, H., and Hein, L. (1993). Heterogeneity of angiotensin synthetic pathways and receptor subtypes: Physiological and pharmacological implications. J. Hypertens. 11:S13-S18.

    Google Scholar 

  • Griendling, K. K., Murphy, T. J., and Alexander, R. W. (1993). Molecular biology of the renin-angiotensin system. Circulation 87:1816–1828.

    Google Scholar 

  • Harada, H., Kitagawa, M., Tanaka, N., Yamamoto, H., Harada, K., Ishihara, M., and Taniguchi, T. (1993). Anti-oncogenic and oncogenic potentials of transcriptional regulators IRF-1 and-2. Science 259:971–974.

    Google Scholar 

  • Hopper, N. M. (1991). Angiotensin converting enzyme: Implications from molecular biology for its physiological functions. Int. J. Biochem. 23:641–647.

    Google Scholar 

  • Horiuchi, M., Koike, G., Yamada, T., Mukoyama, M., Nakajima, M., and Dzau, V. J. (1995). The growth-dependent expression of angiotensin II type 2 receptor is regulated by transcription factors interferon regulatory factor-1 and-2 J. Biol. Chem. 270:20225–20230.

    Google Scholar 

  • Ichiki, T., Kambayashi, Y., and Inagami, T. (1995). Multiple growth factors modulate mRNA expression of angiotensin of angiotensin II type-2 receptor in R3T3 cells. Cir. Res. 77:1070–1076.

    Google Scholar 

  • Inagami, T., and Harris, R. C. (1993). Molecular insights into angiotensin II receptor subtypes. TIPS 8:215–218.

    Google Scholar 

  • Jainchill, J. L., Aaronson, S. A., and Todaro, G. J. (1969). Murine sarcoma and leukemia virus: Assay using clonal lines of contact-inhibited mouse cells. J. Virol. 4:549–553.

    Google Scholar 

  • Kalinec, G., Nazarali, A. J., Hermouet, S., Xu, N., and Gutkind, J. S., (1992). Mutated Ó subunit of the Gq protein induces malignant transformation in NIH 3T3 cells. Mol. Cell. Biol. 12:4687–4693.

    Google Scholar 

  • Kambayashi, Y., Bardhan, S., and Inagami, T. (1993a). Peptide growth factors markedly decrease the ligand binding of angiotensin II type 2 receptor in rat cultured vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 194:478–482.

    Google Scholar 

  • Kambayashi, Y., Bardhan, S., Takahashi, K., Tsuzuki, S., Inui, H., Hamakubo, T., and Inagami, T. (1993b). Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J. Biol. Chem. 268:24543–24546.

    Google Scholar 

  • Krulewitz, A. H., Baur, W. E., and Fanburg, B. L. (1984). Hormonal influence on endothelial cell angiotensin-converting enzyme activity. Am. J. Physiol. 247:C163-C168.

    Google Scholar 

  • Leung, K. H., Roscoe, A., Smith, R. D., Timmermans, P. B. M. W. M., and Chiu, A. T. (1992). Characterization of biochemical response of angiotensin II (AT2) binding sites in the rat pheochromocytoma PC12W cells. Eur. J. Pharmacol. 227:63–67.

    Google Scholar 

  • Michels, K. M., Heemskerk, F. M. J., and Saavedra, J. M. (1993). Selective changes in angiotensin II AT1 and AT2 receptor subtypes in the rat superior colliculus following eye enucleation. Neuroscience 58:835–844.

    Google Scholar 

  • Mukoyama, M., Nakajima, M., Horiuchi, M., Sasamura, H., Pratt, R. E., and Dzau, V. J. (1993). Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven transmembrane receptors. J. Biol. Chem. 268:24539–24542.

    Google Scholar 

  • Munson, P. J. (1983). LIGAND: A computerized analysis of ligand binding data. In Methods in Enzymology, (Langone, J. J., and Van Vunakis, H., eds.), Academic Press, New York, Vol. 92, Part E, pp. 543–576. 1983.

    Google Scholar 

  • Nahmias, C., and Strosberg, A. D. (1995). The angiotensin AT2 receptor: Searching for signal-transduction pathways and physiological function. Trends Pharmacol. Sci. 16:223–225.

    Google Scholar 

  • Nunes-Mamede, M. L., De Mello, F. G., and Martins, A. R. (1990). In ovo and in culture development of chick retinal angiotensin converting enzyme. Neurosci. Lett. 109:174–179.

    Google Scholar 

  • Powell, J., Clozel, J. P., Muller, R. K. M., Kuhn, H., Hefti, F., Hosang, M., and Baumgartner, H. R. (1989). Inhibitors of angiotensin-converting enzyme prevent myointimal proliferation after vascular injury. Science 245:186–188.

    Google Scholar 

  • Pucell, A. G., Hodges, J. C., Sen, I., Bumpus, F. M., and Husain, A. (1991). Biochemical properties of the ovarian granulosa cell type 2-angiotensin II receptor. Endocrinology 128:1947–1959.

    Google Scholar 

  • Rorhbach, M. S. (1978). [Glycine–1–14]hippuryl-histidyl-L-leucine: A substrate for the radiochemical assay of angiotensin-converting-enzyme. Anal. Biochem. 84:272–276.

    Google Scholar 

  • Saavedra, J. M. (1992). Brain and pituitary angiotensin. Endocr. Rev. 13:329–380.

    Google Scholar 

  • Schelling, P., Fischer, H., and Ganten, D. (1991). Angiotensin and cell growth: A link to cardiovascular hypertrophy. J. Hypertens. 9:3–15.

    Google Scholar 

  • Shai, S.-Y., Fishel, R. S., Martin, B. M., Berk, B. C., and Bernstein, K. E. (1992). Bovine angiotensin converting enzyme cDNA cloning and regulation. Circ. Res. 70:1274–1281.

    Google Scholar 

  • Smith, R. D. (1993). Identification of AT2 receptors on Swiss 3T3 cells. Biochem. Mol. Biol. Int. 31:975–982.

    Google Scholar 

  • Stoll, M., Muscha Steckelings, U., Paul, M., Bottari, S. P., Metzger, R., and Unger, T. (1995). The angiotensin AT2-receptor medites inhibition of cell proliferation in coronary endothelial cells. J. Clin. Invest. 95:651–657.

    Google Scholar 

  • Tanaka, N., Ishihara, M., Kitagawa, M., Harada, H., Kimura, T., Matsuyama, T., Lamphier, M. S., Aizawa, S., Mak, T. W., and Taniguchi, T. (1994). Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1. Cell 77:829–839.

    Google Scholar 

  • Tang, S.-S., Rogg, H., Schumacher, R., and Dzau, V. J. (1992). Characterization of nuclear angiotensin II binding sites in rat liver and comparison with plasma membrane receptors. Endocrinology 131:374–380.

    Google Scholar 

  • Timmermans, P. B. M. W. M., Wong, P. C., Chiu, A. T., Herblin, W. F., Benfield, P., Carini, D. J., Lee, R. J., Wexler, R. R., Saye, J. A. M., and Smith, R. D. (1993). Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol. Rev. 45:205–251.

    Google Scholar 

  • Tsutsumi, K., and Saavedra, J. M. (1992). Heterogeneity of angiotensin II AT2 receptors in the rat brain. Mol. Pharmacol. 41:290–297.

    Google Scholar 

  • Whitebread, S., Mele, M., Kamber, B., and de Gasparo, M. (1989). Preliminary biochemical characterization of two angiotensin receptor subtypes. Biochem. Biophys. Res. Commun. 163:284–291.

    Google Scholar 

  • Yang, H. Y. T., and Neff, N. H. (1973). Differential distribution of angiotensin-converting enzyme in the anterior and posterior lobe of the rat pituitary. J. Neurochem. 21:1035–1036.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Section on Pharmacology, National Institute of Mental Health, 10 Center Drive, Building 10/2D57, Bethesda, Maryland, 20892-1264

    Frank M. J. Heemskerk, Stefan Zorad & Juan M. Saavedra

  2. Laboratory of Cellular Development and Oncology, National Institute of Dental Research, Bethesda, Maryland, 20892

    Ningzhi Xu & Silvio J. Gutkind

Authors
  1. Frank M. J. Heemskerk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefan Zorad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ningzhi Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Silvio J. Gutkind
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Juan M. Saavedra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Juan M. Saavedra.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Heemskerk, F.M.J., Zorad, S., Xu, N. et al. Characterization of AT2 Receptor Expression in NIH 3T3 Fibroblasts. Cell Mol Neurobiol 19, 277–288 (1999). https://doi.org/10.1023/A:1006985329240

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1006985329240

Search

Navigation