A great variety of functional assays are in use to evaluate novel ligands of tachykinin receptors. These techniques, however, are generally labor and time consuming and hardly suitable for high throughput screening. Using human recombinant NK-2 receptors expressed in CHO cells, we have developed an assay based on direct measurement of guanine nucleotide exchange on G-proteins. [35S]GTPγS, which cannot be hydrolyzed by the intrinsic GTPase activity of the α-subunit of heterotrimeric G-protein, was employed. Six ligands of NK-2 receptors including both natural neurokinins and neurokinin A analogs were used for the assay validation. The assay was shown to discriminate successfully between partial and full agonism, as well as between agonism and antagonism of the peptides. Besides the exquisite specificity and high throughput, the assay obviates the need for laboratory animals and cumbersome muscle-bath techniques for conventional tachykinin pharmacology. The functional GTPγS assay is a specific and reliable high throughput screening tool for the investigation of pharmacologic properties of neurokinin A analogs. The assay has multiple advantages over existing techniques and is favored for studies on structure-activity relationships in peptides.
References
1.
Krause, J.M., Hershey, A.D., Dykema, P.E., Takeda, Y. (1990). Molecular biological studies on the diversity of chemical signaling in tachykinin peptidergic neurons. Ann. N.Y. Acad. Sci.579:254.
2.
Maggi, C.A. (1995). The mammalian tachykinin receptors. Gen. Pharmacol.26:911.
3.
Nakanishi, S. (1991). Mammalian tachykinin receptors. Ann. Rev. Neurosci.14:123.
4.
Khawaja, A.M., Rogers, D.F. (1996). Tachykinins: Receptor to effector. Int. J. Biochem. Cell Biol.28:721.
5.
Garland, A.M., Grady, E.F., Lovett, M., Vigna, S.R., Frucht, M.M., Krause, E.E., Bunnett, N.W. (1996). Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors. Mol. Pharmacol.49:438.
6.
Schreurs, J., Yamamoto, R., Lyons, J., Munemitsu, S., Conroy, L., Clark, R., Takeda, Y., Krause, J.E., Inns, M. (1995). Functional wild-type and carboxy-terminal-tagged rat substance P receptors expressed in baculovirus-infected insect Sf9 cells. J. Neurochem.64:1622.
7.
Nakajima, Y., Tsuchida, K., Negishi, M., Ito, S., Nakanishi, S. (1992). Direct linkage of the three tachykinin receptors to stimulation of both phosphatidylinositole hydrolysis and cyclic AMP cascade in transfected Chinese hamster ovary cells. J. Biol. Chem.267:2437.
8.
Otsuka, M., Yoshioka, K. (1993). Neurotransmitter functions of mammalian tachykinins. Physiol. Rev.73:229.
9.
Zagorodnyuk, V., Santicioli, P., Turini, D., Maggi, C.A. (1997). Tachykinin NK1 and NK2 receptors mediate non-adrenergic non-cholinergic excitatory neuromuscular transmission in human ileum. Neuropeptides. 31:265.
10.
Zagorodnyuk, V., Maggi, C.A. (1997). Tachykinin NK1 and NK2 receptors mediate non-adrenergic non-cholinergic excitatory neuromuscular transmission in the guinea-pig stomach. Neuroscience. 80:625.
11.
Maggi, C.A., Giuliani, S., Zagorodnyuk, V. (1997). Sequential activation of the triple excitatory transmission to the circular muscle of guinea-pig colon. Neuroscience. 79:263.
12.
Osakada, F., Kubo, K., Goto, K., Kanazava, I., Mukekata, E. (1986). The contractile activities of neurokinin A, B and related peptides on smooth muscles. Eur. J. Pharmacol.120:201.
13.
Erspamer, V., Melchiorri, P. (1973) Active polypeptides of the amphibian skin and their synthetic analogs. Pure Appl. Chem.35:463.
14.
Dion, S., Rouissi, N., Nantel, F., Jkic D., Rhaleb, N.E., Tousignant C., Telemaque, S., Drapeau, G., Regoli, D., Naline E, Advenier, C., Rovero, P., Maggi, C.A. (1990). Structure-activity study of neurokinins: Antagonists for the neurokinin-2 receptor. Pharmacology. 41:184.
15.
Maggi, C.A. (1997). The effects of tachykinins on inflammatory and immune cells. Reg. Peptides.70:75.
16.
Sharif, M.S., Sharif, T.R., Dilling, M.B., Hosoi, H., Lawrence, J.C., Houghton, P. (1997). Rapamycin inhibits substance P-induced protein synthesis and phosphorylation of PHAS-I (4E-BP1) and p70S6K kinase (p70S6K) in human astrocytoma cells. Int. J. Oncol.11:797.
17.
Luo, W., Sharif, T.R., Sharif, M. (1996). Substance P-induced mitogenesis in human astrocytoma cells correlates with activation of the mitogen-activated protein kinase signaling pathway. Cancer Res.56:4983.
18.
Seckl, M.J., Higging, T., Widmer, F., Rozengurt, E. (1997). [D-Arg1,D-Trp5,7,9, Leu11]substance P: A novel potent inhibitor of signal transduction and growth in vitro and in vivo in small cell lung cancer cells. Cancer Res.57:51.
19.
Hardebo, J.E. (1985). Substance P and the pathogenesis of cluster headache. In Tachykinin Antagonists. R. Håkanson and F. Sandler (eds.), pp. 71-80. Elsevier, New York.
20.
Toru, M.S., Watanabe, H., Shibuya, T., Nishikawa, K., Noda, H., Mitsushio, H., Ichikawa, A., Kurumaji, M., Takashima, N., Mataga, N., Ogawa, A. (1988). Neurotransmitters, receptors and neuropeptides in post-mortem brains of chronic schizophrenic patients. Acta Psychiatr. Scand.78:121.
21.
Beal, M.F., Mazurek, M.F. (1987). Substance P-like immunoreactivity is reduced in Alzheimer's disease cerebral cortex. Neurology. 37:1205.
22.
Mauborgne, A., Javoy-Agid, F., Legrand, J.C., Agid, Y., M., Cesselin, F. (1983). Decrease of substance P-like immunoreactivity in the substantia nigra and pallidum of parkinsonian brains. Brain Res.268:167.
23.
DeLaet, M.H., Dassonville, M., Steyaert, H., Vanderveken, E., Lotstra, F., Vierendeels, G., Przedborski, S., Vaudry, H., Vanderhaeghen, J.J. (1989). Decrease of vasoactive intestinal peptide, methionine-enkephalin, substance P and increase of neuropeptide Y immunoreactive nerve fibers in angangliotic colon of Hirschsprung's disease. Neurochem. Int.14:135.
24.
Arai, H., Emson, P.C., Carrasco, L.H. (1987). Huntington's disease: Changes in tachykinin content in post mortem brains. Ann. Neurol.22:587.
25.
Mapp, P.I., Kidd, B.L., Gibbson, S.G., Terry, J.M., Revell, P.A., Ibrahim, N.B.N., Blake, D.R., Polak, J.M. (1990). Substance P-, calcitonin gene-related peptide- and C-flanking peptide of neuropeptide Y-immunoreactive fibers are present in normal synovium but depleted in patients with rheumatoid arthritis. Neuroscience. 37:143.
26.
Barnes, P.J. (1987). Airway neuropeptides and asthma. Trends Pharmacol. Sci.8:24.
27.
Lowe, J.A. (1996). Nonpeptide tachykinin antagonists: Medicinal chemistry and molecular biology. Med. Res. Rev.16:527.
28.
Regoli, D., Drapeau, G., Dion, S., D'Orleans-Juste, P. (1989). Receptors for substance P and related neurokinins. Pharmacology. 38:1.
29.
Regoli, D., Nguyen, Q.T., Jukie, D. (1994). Neurokinin receptors subtypes characterized by biological assays. Life Sci.54:2035.
30.
Regoli, D., Drapeau, G., Dion, S., D'Orleans-Juste, P. (1987). Receptors for substance P and neurokinins. Life Sci.40:109.
31.
Regoli, D., Cadieux, A., D'Orleans-Juste, P. (1993). Vasoactive peptides and their receptors. Methods Neurosci.11:43.
32.
Murnin, M., Lovas, S., Allen, J.M., Murphy, R.F. (1997). Organ/tissue preparations for the assessment of agonist/antagonist activity. In Methods in Molecular Biology, Neuropeptide Protocols. G.B. Irvine and C.H. Williams (eds.), pp. 343-352. Humana Press, Totowa, NJ.
33.
Stratowa, C., Machat, H., Bürger, E., Himmler, A., Schäfer, R., Speval, W., Weyer, U., Wiche-Castanon, M., Czernilofsky, A.P. (1995). Functional characterization of the human neurokinin receptors NK1, NK2, and NK3 based on a cellular assay system. J. Receptor Signal Transd. Res.15:617.
34.
Sundelin, J.B., Provvedini, D.M., Wahlesttedt, C.R., Laurell, H., Pohl, J.S., Peterson P.A. (1992). Molecular cloning of the murine substance K and substance P receptor genes. Eur. J. Biochem.203:625.
35.
Messier, T.L., Dorman, C.M., Braüner-Osborne, H., Eubanks, D., Brann, M.R. (1995). High throughput assays of cloned adrenergic, muscarinic, neurokinin, and neurotrophin receptors in living mammalian cells. Pharmacol. Toxicol.76:308.
36.
Brann, M., Messier, T., Dorman, C., Lannigan, D. (1996). Cell-based assays for G-protein-coupled/tyrosine kinase-coupled receptors. J. Biomol. Screening. 1:43.
37.
Gilman, A.G. (1987). G proteins: Transducers of receptor-generated signals. Ann. Rev. Biochem.56:615.
38.
Lazareno, S., Farries, T., Birdsall, N.J.M. (1993). Pharmacological characterization of guanine nucleotide exchange reactions in membranes from CHO cells stably transfected with human muscarinic receptors M1-M4. Life Sci.52:449.
39.
Lorenzen, A., Fuss, M., Vogt, H., Schwabe, U. (1993). Measurement of guanine nucleotide-binding protein activation by A1 adenosine receptor agonists in bovine brain membranes: Stimulation of guanosine-5′-O-(3-[35S]thio)-triphosphate binding. Mol. Pharmacol.44:115.
40.
Traynor, J.R., Nahorski, S.R. (1995). Modulation by μ-opioid agonists of guanosine-5′-O-(3-[35S]thio)-triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. Mol. Pharmacol.47:848.
41.
Gardner, B., Hall, D.A., Strange, P.G. (1996). Pharmacological analysis of dopamine stimulation of [35S]-GTPγS binding via human D2short and D2long dopamine receptors expressed in recombinant cells. Br. J. Pharmacol.118:1544.
42.
Spencer, C.J., Nelson, C.B., Schwarz, R.D. (1996). GTPγS binding as a tool to characterize metabotropic glutamate receptor agonists in CHO cells expressing subtypes 2 and 6. Soc. Neurosci. Abstr.22:412.4.
43.
Pauwels, P.J., Tardiff, S., Palmier, C., Wurch, T., Colpaert, F.C. (1997). How efficacious are 5-HT1B/D receptor ligands: An answer from GTPγS binding studies with stably transfected C6-glial cell lines. Neuropharmacology. 36:499.
44.
Ashkenazi, A., Peralta, E.G., Winslow, J.W., Ramachandran, J., Capon, D.J. (1989). Functionally distinct G proteins selectively couple different receptors to PI hydrolysis in the same cell. Cell. 56:487.
45.
Rovero, S., Pestellini, V., Rhaleb, N.E., Dion, S., Rouissi, N., Tousignant, C., Telemaque, S., Drapeau, G., Regoli, D. (1989). Structure-activity studies of neurokinin A. Neuropeptides.13:263.
46.
Ötvös, F.L., Gembitsky, D.S., Murphy, R.F., Lovas, S. (1998). Neurokinin A, analogs with conformational constraint. Biochem. Soc. Trans.26(suppl):3.
47.
Waeber, C., Moskowitz, M.A. (1997). 5-Hydroxytryptamine1A and 5-hydroxytryptamine1B receptors stimulate [35S]guanosine-5′-O-(3-thio)triphosphate binding to rodent brain sections as visualized by in vitro autoradiography. Mol. Pharmacol.52:623.
48.
Wieland, T., Jakobs, K.H. (1994). Receptor-stimulated guanosine-5′-O(γ-thio)triphosphate binding by G proteins. Methods Enzymol.237:3.
49.
Fong, T.M., Yu, H., Strader, C.D. (1992). Molecular basis for species selectivity of the neurokinin-1 receptor antagonists CP-96,345 and RP67580. J. Biol. Chem.267:25668.
50.
D'Orleans-Juste, P., Dion, S., Drapeau, G., Regoli, D. (1986). Different receptors are involved in the endothelium-mediated relaxation and the smooth muscle contraction of the rabbit pulmonary artery in response to substance P and related neurokinins. Eur. J. Pharmacol.125:37.
51.
Bräuner-Osborn, H., Brann, M.R. (1996). Pharmacology of muscarinic acetylcholine receptor subtypes (m1-m5): High throughout assays in mammalian cells. Eur. J. Pharmacol.295:93.
