Summary
Peptides labelled with the fluorophore cyanine 3 were used to study naturally expressed neuropeptide receptors by confocal microscopy in continuous cell lines, primary cultures, and unfixed tissue. Swiss 3T3 fibroblasts bound cyanine 3-gastrin-releasing peptide at 4°C, and internalized the peptide after 10 min at 37°C. Internalization was specific, since it was blocked by incubation with unlabelled peptide. Primary cultures of myenteric neurons of the guinea pig incubated with cyanine 3-substance P at 4°C had specific surface labelling. After 30 s at 37°C, the peptide was internalized into vesicles in both the soma and neurites. Direct observation of live neurons showed movement of fluorescent vesicles to a perinuclear region after 30 min. Endocytosis was associated with a loss of surface binding sites. Unfixed whole mounts of guinea pig and rat ileum were incubated with cyanine 3-neurokinin A at 4°C. After 5 min at 37°C, Cy3-neurokinin A was specifically internalized in neurons and smooth muscle cells. After 30 min, a perinuclear labelling occurred in some cells. Labelling in rat neurons was diminished by the NK3-R antagonist SR142801. Thus, cyanine 3-neuropeptides are valuable tools to study expression and endocytosis of naturally expressed receptors.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Able, M. E., Lee, J. C. &Rosenau, W. (1970) Lymphocyte-target cell interactionin vitro.Am. J. Pathol. 60, 421–34.
Anderson, K. M. &Peach, M. J. (1994) Receptor binding and internalization of a unique biologically active angiotensin II-colloidal gold conjugate: morphological analysis of angiotensin II processing in isolated vascular strips.J. Vasc. Res. 31, 10–17.
Anion, P. A., Reeve, J. R., Jr., Rivier, J. E., Vidrich, A., Schepp, W. &Shanahan, F. (1991) Biotinylation of a bombesin/gastrin-releasing peptide analogue for use as a receptor probe.Peptides 12, 375–81.
Balice-Gordon, R. J. &Lichtman, J. W. (1994) Long-term synapse loss induced by focal blockade of postsynaptic receptors.Nature 372, 519–24.
Böhm, S., Khitin, L. M., Grady, E. F., Aponte, G., Payan, D. G., &Bunnett, N. W. (1996a) Mechanisms of desensitization and resensitization of proteinase-activated receptor-2.J. Biol. Chem. 271, 22003–16.
Böhm, S., Grady, E. F., Smeekens, S. P., Payan, D. G. & Bunnett, N. W. (1996b) Identification of tyrosine-containing endocytic motifs in the carboxyl-tail and seventh transmembrane domain domains of the NK1 receptor.J. Biol. Chem. Submitted.
Bowden, J. J., Garland, A. M., Baluk, P., Lefevre, P., Grady, E. F., Vigna, S. R., Bunnett, N. W. &Mcdonald, D. M. (1994) Direct observation of substance P-induced internalization of neurokinin 1 (NK1) receptors at sites of inflammation.Proc. Natl. Acad. Sci. USA 91, 8964–8.
Bunnett, N. W., Dazin, P. F., Payan, D. G. &Grady, E. F. (1995) Characterization of receptors using cyanine 3-labeled neuropeptides.Peptides 16, 733–40.
Burgevin, M. C., Castel, M. N., Quarteronet, D., Chevet, T. &Laduron, P. M. (1992) Neurotensin increases tyrosine hydroxylase messenger RNA-positive neurons in substantia nigra after retrograde axonal transport.Neuroscience 49, 627–33.
Castel, M. N., Malgouris, C., Blanchard, J. C. &Laduron, P. M. (1990) Retrograde axonal transport of neurotensin in the dopaminergic nigrostriatal pathway in the rat.Neuroscience 36, 425–30.
Faure, M.-P., Gaudreau, P., Shaw, I., Cashman, N. R. &Beaudet, A. (1994) Synthesis of biologically active fluorescent probe for labeling neurotensin receptors.J. Histochem. Cytochem. 42, 755–63.
Faure, M.-P., Alonso, A., Nouel, D., Gaudriault, G., Dennis, M., Vincent, J.-P. &Beaudet, A. (1995) Somatodendritic internalization and perinuclear targeting of neurotensin in the mammalian brain.J. Neurosci. 15, 4140–7.
Garland, A. M., Grady, E. F., Payan, D. G., Vigna, S. R. &Bunnett, N. W. (1994) Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells.Biochem. J. 303, 177–86.
Garland, A. M., Grady, E. F., Lovett, M., Vigna, S. R., Frucht, M. M., Krause, J. E. &Bunnett, N. W. (1996) Mechanisms of desensitization and resensitization of the G-protein coupled NK-1 and NK-2 receptors.J. Mol. Pharm. 49, 438–46.
Grady, E. F., Sechi, L. A., Griffin, C. A., Schambelan, M. &Kalinyak, J. E. (1991) Expression of AT2 receptors in the developing rat fetus.J. Clin. Invest. 88, 921–33.
Grady, E. F., Garland, A. G., Gamp, P. D., Lovett, M., Payan, D. G. &Bunnett, N. W. (1995a) Delineation of the endocytic pathway of substance P and the seven transmembrane domain NK1 receptor.Mol. Biol. Cell. 6, 509–24.
Grady, E. F., Slice, L. W., Brant, W. O., Walsh, J. H., Payan, D. G. &Bunnett, N. W. (1995b) Direct observation of endocytosis of gastrin releasing peptide and its receptor.J. Biol. Chem. 270, 4603–11.
Grady, E. F., Baluk, P., Böhm, S., Gamp, P. D., Wong, H., Payan, D. G., Ansel, J., Portbury, A. L., Furness, J. B., McDonald, D. M. & Bunnett, N. W. (1996a) Characterization of antisera specific to NK1, NK2, and NK3 neurokinin receptors and their utilization to localize receptors in the rat gastro-intestinal tract.J Neurosci. in press.
Grady, E. F., Gamp, P. D., Baluk, P., McDonald, D. M., Payan, D. G., & Bunnett, N. W. (1996b) Endocytosis and recycling of NK1 tachykinin receptors in enteric neurons.Neuroscience in press.
Grider, J. R. &Bonilla, O. M. (1994) Differential expression of substance P, somatostatin and VIP in neurons from cultured myenteric ganglia.Am. J. Physiol. 267, G322–7.
Herkenham, M. &Pert, C. B. (1982) Light microscopic localization of brain opiate receptors: a general autoradiographic method which preserves tissue quality.J. Neurosci. 2, 1129–49.
Liu, H., Brown, J. L., Jasmin, L., Magio, J. E., Vigna, S. R., Mantyh, P. W. &Basbaum, A. I. (1994) Synaptic relationship between substance P and the substance P receptor: light and electron microscopic characterization of the mismatch between neuropeptides and their receptors.Proc. Natl. Acad. Sci. USA 91, 1009–13.
Mantyh, P. W., Gates, T., Mantyh, C. R. &Maggio, J. E. (1989) Autoradiographic localization and characterization of tachykinin receptor binding sites in the rat brain and peripheral tissues.J. Neurosci. 9, 258–79.
Mantyh, P. W., Allen, C. J., Ghilardi, J. R., Rogers, S. D., Mantyh, C. R., Liu, H., Basbaum, A. I., Vigna, S. R. &Maggio, J. E. (1995a) Rapid endocytosis of a G protein-coupled receptor: Substance P-evoked internalization of its receptor in the rat striatum in vivo.Proc. Natl. Acad. Sci. USA 92, 2622–6.
Mantyh, P. W., Demaster E., Malhotra, A., Ghilardi, J. R., Rogers, S. D., Mantyh, C. R., Liu, H., Basbaum, A. I., Vigna, S. R. &Maggio, J. E. (1995b) Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation.Science 268, 1629–32.
Overly, C. C., Lee, K-D., Berthiaume, E. &Hollenbeck, P. J. (1995) Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine.Proc. Natl. Acad. Sci. USA 92, 3156–60.
Patacchini, R. &Maggi, C. A. (1995) Tachykinin receptors and receptor subtypes.Arch. Int. Pharmacodyn. Ther. 329, 161–84.
Roettger, B. F., Rentsch, R. U., Pinon, D., Holicky, E., Hadac, E. M., Larkin, J. M. &Miller, L. J. (1995) Dual pathways of internalization of the cholecystokinin receptor.J. Cell Biol. 128, 1029–41.
Russell, S. W., Rosenau, W. &Lee, J. C. (1972) Cytolysis induced by human lymphotoxin.Am. J. Pathol. 69, 103–17.
Sargent, P. B. (1994) Double-label immunofluorescence with the laser scanning confocal microscopy using cyanine dyes.Neuroimage 1, 288–95.
Sechi, L. A., Grady, E. F., Griffin, C. A., Kalinyak, J. E. &Schambelan, M. (1992) Distribution of angiotensin II receptor subtypes in rat and human kidney.Am. J. Physiol. 262, F236–40.
Tidball, J. G., Spencer, M. J. &St. Pierre, B. A. (1992) PDGF-receptor concentration is elevated in regenerative muscle fibers in dystrophin-deficient muscle.Exp. Cell Res. 203, 141–9.
Van Epps, D. E., Simpson, S., Bender, J. G. &Chenoweth, D. E. (1990) Regulation of C5a and formyl peptide receptor expression on human polymorphonuclear leukocytes.J. Immunol. 144, 1062–8.
Van Mier, P. &Lichtman, J. W. (1994) Regenerating muscle fibers induce directional sprouting from nearby nerve terminals: studies in living mice.J. Neurosci. 14, 5672–86.
Vigna, S., Bowden, G., McDonald, D. M., Fisher, J., Okamoto, A., McVey, D. C., Payan, D. &Bunnett, N. W. (1994) Characterization of antibodies to the rat substance P (NK1) receptor and to a chimeric substance P receptor expressed in mammalian cells.J. Neurosci. 14, 834–45.
Von Banchet, G. S. &Heppelmann, B. (1995) Non-radioactive localization of substance P binding sites in rat brain and spinal cord using peptides labeled with 1.4-nm gold particles.J. Histochem. Cytochem. 43, 821–7.
Wessendorf, M. W. &Brelje, T. C. (1992) Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18.Histochemistry 98, 83–8.
Wyllie, A. H., Kerr, J. F. R. &Currie, A. R. (1980) Cell death: the significance of apoptosis.Int. Rev. Cytol. 68, 251–306.
Yau, W. M., Dorsett, J. A. &Parr, E. L. (1989) Characterization of acetylcholine release from enzyme-dissociated myenteric ganglia.Am. J. Physiol. 256, G233–9.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bunnett, N.W., Payan, D.G. & Grady, E.F. Detection of naturally expressed receptors for gastrin-releasing peptide and tachykinins using cyanine 3-labelled neuropeptides. Histochem J 28, 811–826 (1996). https://doi.org/10.1007/BF02272154
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF02272154