radiochemical and fluorescence- fluorescence-based methods
4.4 Summary and conclusionsSummary andconclusions
4.4 Summary and conclusions
HEK293-hH2R-qs5-HA cells are appropriate for the performance of both radioligand and flow cytometric binding assays for the hH2R. The coexpression of the chimera in HEK293-hH2 R-qs5-HA cells did not significantly influence the hH2R binding properties of the investigated ligands.
HEK293-hH2R-qs5-HA cells are appropriate for the performance of both radioligand and flow cytometric binding assays for the hH2R. The coexpression of the chimera in HEK293-hH2 R-qs5-HA cells did not significantly influence the hH2R binding properties of the investigated ligands.
In order to avoid the use of the red diode laser which is quite sensitive with regard to vibration, flow cytometric competition binding experiments with standard ligands for validation were performed with 7, which could be excited with the less susceptible argon laser, indispensible in flow cytometry. Those measurements revealed in part lower affinities for standard ligands in comparison to radioligand binding data determined within the scope of this work or reported in literature, respectively (Table 4.5). Since the use of DMSO was identified as a possible reason for these discrepancies, investigations at reduced concentrations of DMSO are recommended if possible. In case of famotidine and of tiotidine, the omission of DMSO could lead to insufficient solubility. As flow cytometric competition binding experiments revealed an affinity of compound 8 comparable to tiotidine, its radioactive form incorporating a [3H]-propionyl moiety represents a promising radioligand for the hH2R.
In order to avoid the use of the red diode laser which is quite sensitive with regard to vibration, flow cytometric competition binding experiments with standard ligands for validation were performed with 7, which could be excited with the less susceptible argon laser, indispensible in flow cytometry. Those measurements revealed in part lower affinities for standard ligands in comparison to radioligand binding data determined within the scope of this work or reported in literature, respectively (Table 4.5). Since the use of DMSO was identified as a possible reason for these discrepancies, investigations at reduced concentrations of DMSO are recommended if possible. In case of famotidine and of tiotidine, the omission of DMSO could lead to insufficient solubility. As flow cytometric competition binding experiments revealed an affinity of compound 8 comparable to tiotidine, its radioactive form incorporating a [3H]-propionyl moiety represents a promising radioligand for the hH2R.
Table 4.5: Comparison of binding data determined in flow cytometric and radioligand binding experiments on HEK293-hH2R-qs5-HA cells with radioligand binding data reported in literature (Ki
[µM]; mean values ± SEM).
References 89
4.5 References
Arima, N., Yamashita, Y., Nakata, H., Nakamura, A., Kinoshita, Y., Chiba, T., 1991. Presence of histamine H2-receptors on human gastric carcinoma cell line MKN-45 and their increase by retinoic acid treatment. Biochem. Biophys. Res. Commun. 176, 1027-1032.
Bohn, B., 1980. Flow cytometry: A novel approach for the quantitative analysis of receptor-ligand interactions on surfaces of living cells. Mol. Cell. Endocrinol. 20, 1-15.
Buckland, K.F., Williams, T.J., Conroy, D.M., 2003. Histamine induces cytoskeletal changes in human eosinophils via the H4 receptor. Br. J. Pharmacol. 140, 1117-1127.
Burde, R., Seifert, R., 1996. Stimulation of histamine H2- (and H1)-receptors activates Ca2+ influx in all-trans-retinoic acid-differentiated HL-60 cells independently of phospholipase C or adenylyl cyclase. Naunyn-Schmiedeberg's Arch. Pharmacol. 353, 123-129.
Cheng, Y.-C., Prusoff, W.H., 1973. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction.
Biochem. Pharmacol. 22, 3099-3108.
Conklin, B., Herzmark, P., Ishida, S., Voyno-Yasenetskaya, T., Sun, Y., Farfel, Z., Bourne, H., 1996.
Carboxyl-terminal mutations of Gq alpha and Gs alpha that alter the fidelity of receptor activation. Mol. Pharmacol. 50, 885-890.
Conklin, B.R., Farfel, Z., Lustig, K.D., Julius, D., Bourne, H.R., 1993. Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature 363, 274-276.
Edwards, B.S., Oprea, T., Prossnitz, E.R., Sklar, L.A., 2004. Flow cytometry for throughput, high-content screening. Curr. Opin. Chem. Biol. 8, 392-398.
Hofinger, E., 2007. Recombinant Expression, Purification and Characterization of Human Hyaluronidases. In: Doctoral Thesis, University of Regensburg. http://www.opus-bayern.de/uni-regensburg/volltexte/2007/788/.
Hulme, E.C., Birdsall, N.J.M., 1992. Strategy and tactics in receptor-binding studies. In: Receptor-Ligand Interactions: A Practical Approach (Practical Approach Series), Hulme, E.C. (Ed.).
Oxford University Press, New York, pp. 63-176.
Hulme, E.C., Buckley, N.J., 1992. Receptor preparations for binding studies. In: Receptor-Ligand Interactions: A Practical Approach (Practical Approach Series), Hulme, E.C. (Ed.). Oxford University Press, New York, pp. 177-212.
Keen, M., 1995. The problems and pitfalls of radioligand binding. In: Methods in Molecular Biology, Vol. 41: Signal Transduction Protocols, Kendall, D.A., Hill, S.J. (Eds.). Humana Press, Totowa, pp. 1-16.
Kelley, M.T., Bürckstümmer, T., Wenzel-Seifert, K., Dove, S., Buschauer, A., Seifert, R., 2001. Distinct Interaction of Human and Guinea Pig Histamine H2-Receptor with Guanidine-Type Agonists.
Mol. Pharmacol. 60, 1210-1225.
Kozak, M., 1987. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs.
Nucleic Acids Res. 15, 8125-8148.
Kozak, M., 2002. Pushing the limits of the scanning mechanism for initiation of translation. Gene 299, 1-34.
Kracht, J., 2001. Bestimmung der Affinität und Aktivität subtypselektiver Histamin- und Neuropeptid Y-Rezeptorliganden an konventionellen und neuen pharmakologischen In-vitro-Modellen. In:
Doctoral Thesis, University of Regensburg.
Kuckuck, F.W., Edwards, B.S., Sklar, L.A., 2001. High throughput flow cytometry. Cytometry 44, 83-90.
Lazareno, S., 2001. Quantification of receptor interactions using binding methods. J. Recept. Signal Transduct. 21, 139 - 165.
Leurs, R., Smit, M.J., Wiro, M.B.P., Timmerman, H., 1994. Pharmacological characterization of the human histamine H2 receptor stably expressed in Chinese hamster ovary cells. Br. J.
Pharmacol. 112, 847-854.
Mayer, M., 2002. Entwicklung fluorimetrischer Methoden zur Bestimmung der Affinität und Aktivität von Liganden G-Protein-gekoppelter Rezeptoren an intakten Zellen. In: Doctoral Thesis, University of Regensburg. http://www.opus-bayern.de/uni-regensburg/volltexte/2002/71/.
McFarthing, K.G., 1992. Selection and synthesis of receptor-specific radioligands. In: Receptor-Ligand Interactions: A Practical Approach (Practical Approach Series), Hulme, E.C. (Ed.). Oxford University Press, New York, pp. 1-18.
Preuss, H., Ghorai, P., Kraus, A., Dove, S., Buschauer, A., Seifert, R., 2007. Constitutive Activity and Ligand Selectivity of Human, Guinea Pig, Rat, and Canine Histamine H2 Receptors. J.
Pharmacol. Exp. Ther. 321, 983-995.
Ramirez, S., Aiken, C.T., Andrzejewski, B., Sklar, L.A., Edwards, B.S., 2003. High-throughput flow cytometry: Validation in microvolume bioassays. Cytometry A 53A, 55-65.
Schneider, E., 2005. Development of Fluorescence-Based Methods for the Determination of Ligand Affinity, Selectivity and Activity at G-Protein Coupled Receptors. In: Doctoral Thesis, University of Regensburg.
Schneider, E., Mayer, M., Ziemek, R., Li, L., Hutzler, C., Bernhardt, G., Buschauer, A., 2006. A Simple and Powerful Flow Cytometric Method for the Simultaneous Determination of Multiple Parameters at G Protein-Coupled Receptor Subtypes. ChemBioChem 7, 1400-1409.
Seifert, R., Wenzel-Seifert, K., Bürckstümmer, T., Pertz, H.H., Schunack, W., Dove, S., Buschauer, A., Elz, S., 2003. Multiple Differences in Agonist and Antagonist Pharmacology between Human and Guinea Pig Histamine H1-Receptor. J. Pharmacol. Exp. Ther. 305, 1104-1115.
Thomas, P., Smart, T.G., 2005. HEK293 cell line: A vehicle for the expression of recombinant proteins.
J. Pharmacol. Toxicol. Methods 51, 187-200.
Wang, J.-X., Yamamura, H.I., Wang, W., Roeske, W.R., 1992. The use of the filtration technique in in vitro radioligand binding assays for membrane-bound and solubilized receptors. In: Receptor-Ligand Interactions: A Practical Approach (Practical Approach Series), Hulme, E.C. (Ed.).
Oxford University Press, New York, pp. 213-234.
Wood, M., Chaubey, M., Atkinson, P., Thomas, D.R., 2000. Antagonist activity of meta-chlorophenylpiperazine and partial agonist activity of 8-OH-DPAT at the 5-HT7 receptor. Eur.
J. Pharmacol. 396, 1-8.
Wurm, F., Bernard, A., 1999. Large-scale transient expression in mammalian cells for recombinant protein production. Curr. Opin. Biotechnol. 10, 156-159.
Xie, S.-X., Ghorai, P., Ye, Q.-Z., Buschauer, A., Seifert, R., 2006. Probing Ligand-Specific Histamine H1- and H2-Receptor Conformations with NG-Acylated Imidazolylpropylguanidines. J.
Pharmacol. Exp. Ther. 317, 139-146.
Young, S.M., Bologa, C.M., Fara, D., Bryant, B.K., Strouse, J.J., Arterburn, J.B., Ye, R.D., Oprea, T.I., Prossnitz, E.R., Sklar, L.A., et al., 2009. Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes. Cytometry A 75A, 253-263.
Ziemek, R., 2006. Development of binding and functional assays for the neuropeptide Y Y2 and Y4
receptors. In: Doctoral Thesis, University of Regensburg. http://www.opus-bayern.de/uni-regensburg/volltexte/2006/679/.
Ziemek, R., Brennauer, A., Schneider, E., Cabrele, C., Beck-Sickinger, A.G., Bernhardt, G., Buschauer, A., 2006. Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands. Eur. J. Pharmacol. 551, 10-18.
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