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. 1995 Nov;116(6):2715–2723. doi: 10.1111/j.1476-5381.1995.tb17232.x

Characteristics of the binding of [3H]-mepyramine to intact human U373 MG astrocytoma cells: evidence for histamine-induced H1-receptor internalisation.

S Hishinuma 1, J M Young 1
PMCID: PMC1909113  PMID: 8590995

Abstract

1. The kinetics of the binding of 5 nM [3H]-mepyramine to sites on intact human U373 MG astrocytoma cells, sensitive to inhibition by 2 microM pirdonium, were temperature-dependent. At 37 degrees C the half-time for association was 0.9 +/- 0.4 min and at 4 degrees C 19 +/- 3 min. Dissociation of bound [3H]-mepyramine was fast at 37 degrees C, t0.5 1.5 +/- 0.3 min, but at 6 degrees C dissociation initiated by dilution or addition of unlabelled mepyramine was negligible over 120 min. The very slow dissociation at 6 degrees C made it possible to reduce the level of pirdonium-insensitive binding from 56 +/- 5% to 39 +/- 5% by washing the cells in ice-cold medium before filtration. 2. The binding of [3H]-mepyramine sensitive to 2 microM temelastine, measured after 10 min equilibration at 37 degrees C, failed to saturate and was resolved into an hyperbola and an apparently linear component, whereas the fit to the binding of [3H]-mepyramine sensitive to 2 microM pirdonium was not significantly improved over that to an hyperbola. The mean Kd for the binding of [3H]-mepyramine to the saturable component, 2.5 +/- 0.4 nM, was in close agreement with the value of 3.5 nM for mepyramine derived from inhibition of histamine H1-receptor-mediated inositol phosphate formation in U373 MG cells. 3. Curves for the inhibition of the binding of 5 nM [3H]-mepyramine to U373 MG cells by histamine H1-receptor antagonists were biphasic and were fitted to a two site-model. Affinities calculated from the best-fit IC50 values for the high-affinity site correlated well with those expected for binding to H1-receptors. 4. The percentages of the high-affinity site in curves of the inhibition of [3H]-mepyramine binding to intact U373 MG cells by two tertiary amine antagonists, norpirdonium and 4-methyldiphenhydramine, 68 +/- 3 and 63 +/- 4%, were significantly greater than the percentages of the high-affinity site in the inhibition curves of their quaternary derivatives, 50 +/- 1 and 45 +/- 3%, respectively. Similarly, the percentage of the high-affinity site for unlabelled mepyramine, 65 +/- 7%, was greater than for the non-cell penetrant H1-antagonist temelastine, 42 +/- 5%. 5. Incubation of U373 MG cells with 100 microM histamine at 37 degrees C, followed by washing twice in ice-cold medium and then incubation with 1-15 nM [3H]-mepyramine for 120 min at 4 degrees C, resulted in a decrease in the binding of [3H]-mepyramine sensitive to 2 microM pirdonium, compared to control cells not exposed to histamine. The binding of [3H]-mepyramine in the absence of pirdonium was not altered by histamine pretreatment, whereas the level of the pirdonium-insensitive binding was significantly increased, except after 1 min exposure to histamine. The decreases in the pirdonium-sensitive binding after 5, 10 and 60 min incubation with 100 microM histamine were 41 +/- 6, 56 +/- 6 and 67 +/- 8%, respectively, but the decrease after 1 min incubation with histamine, 16 +/- 8%, was not statistically significant. 6. The results are consistent with the binding of [3H]-mepyramine to intact U373 MG cells being to both plasma membrane and intracellular histamine H1-receptors. The high-affinity binding sensitive to the non-cell penetrant quaternary compounds and to temelastine is thus to plasma membrane H1-receptors. On exposure to 100 microM histamine receptors are translocated to the intracellular pool, since the change in the high-affinity binding of [3H]-mepyramine is primarily in the level of the pirdonium-insensitive binding, rather than in the total binding.

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Selected References

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  1. Arias-Montaño J. A., Berger V., Young J. M. Calcium-dependence of histamine- and carbachol-induced inositol phosphate formation in human U373 MG astrocytoma cells: comparison with HeLa cells and brain slices. Br J Pharmacol. 1994 Feb;111(2):598–608. doi: 10.1111/j.1476-5381.1994.tb14779.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arias-Montaño J. A., Young J. M. Characteristics of histamine H1 receptors on HeLa cells. Eur J Pharmacol. 1993 May 15;245(3):291–295. doi: 10.1016/0922-4106(93)90110-u. [DOI] [PubMed] [Google Scholar]
  3. Barsoum G. S., Gaddum J. H. The pharmacological estimation of adenosine and histamine in blood. J Physiol. 1935 Aug 22;85(1):1–14. doi: 10.1113/jphysiol.1935.sp003298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bloemers S. M., Leurs R., Smit M. J., Verheule S., Tertoolen L. G., Timmerman H., de Laat S. W. Mouse P19 embryonal carcinoma cells express functional histamine H1-receptors. Biochem Biophys Res Commun. 1993 Feb 26;191(1):118–125. doi: 10.1006/bbrc.1993.1192. [DOI] [PubMed] [Google Scholar]
  5. Bristow D. R., Banford P. C., Bajusz I., Vedat A., Young J. M. Desensitization of histamine H1 receptor-mediated inositol phosphate accumulation in guinea pig cerebral cortex slices. Br J Pharmacol. 1993 Sep;110(1):269–274. doi: 10.1111/j.1476-5381.1993.tb13804.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bristow D. R., Zamani M. R. Desensitization of histamine H1 receptor-mediated inositol phosphate production in HeLa cells. Br J Pharmacol. 1993 Jun;109(2):353–359. doi: 10.1111/j.1476-5381.1993.tb13577.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown R. D., Prendiville P., Cain C. Alpha 1-adrenergic and H1-histamine receptor control of intracellular Ca2+ in a muscle cell line: the influence of prior agonist exposure on receptor responsiveness. Mol Pharmacol. 1986 Jun;29(6):531–539. [PubMed] [Google Scholar]
  8. Calcutt C. R., Ganellin C. R., Jackson B., Leigh B. K., Owen D. A., Smith I. R. Evidence for low brain penetration by the H1-receptor antagonist temelastine (SK&F 93944). Eur J Pharmacol. 1987 Jan 6;133(1):65–74. doi: 10.1016/0014-2999(87)90206-8. [DOI] [PubMed] [Google Scholar]
  9. Chang R. S., Tran V. T., Snyder S. H. Characteristics of histamine H1-receptors in peripheral tissues labeled with [3H]mepyramine. J Pharmacol Exp Ther. 1979 Jun;209(3):437–442. [PubMed] [Google Scholar]
  10. Cioffi C. L., el-Fakahany E. E. Differential sensitivity of phosphoinositide and cyclic GMP responses to short-term regulation by a muscarinic agonist in mouse neuroblastoma cells. Correlation with down-regulation of cell surface receptors. Biochem Pharmacol. 1989 Jun 1;38(11):1827–1834. doi: 10.1016/0006-2952(89)90418-8. [DOI] [PubMed] [Google Scholar]
  11. Cowlen M. S., Barnes M. R., Toews M. L. Regulation of histamine H1 receptor-mediated phosphoinositide hydrolysis by histamine and phorbol esters in DDT1 MF-2 cells. Eur J Pharmacol. 1990 Mar 13;188(2-3):105–112. doi: 10.1016/0922-4106(90)90045-y. [DOI] [PubMed] [Google Scholar]
  12. Dickenson J. M., Hill S. J. Homologous and heterologous desensitization of histamine H1- and ATP-receptors in the smooth muscle cell line, DDT1MF-2: the role of protein kinase C. Br J Pharmacol. 1993 Dec;110(4):1449–1456. doi: 10.1111/j.1476-5381.1993.tb13984.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dillon-Carter O., Chuang D. M. Homologous desensitization of muscarinic cholinergic, histaminergic, adrenergic, and serotonergic receptors coupled to phospholipase C in cerebellar granule cells. J Neurochem. 1989 Feb;52(2):598–603. doi: 10.1111/j.1471-4159.1989.tb09161.x. [DOI] [PubMed] [Google Scholar]
  14. Galper J. B., Dziekan L. C., O'Hara D. S., Smith T. W. The biphasic response of muscarinic cholinergic receptors in cultured heart cells to agonists. Effects on receptor number and affinity in intact cells and homogenates. J Biol Chem. 1982 Sep 10;257(17):10344–10356. [PubMed] [Google Scholar]
  15. Gibson W. J., Roques T. W., Young J. M. Modulation of antagonist binding to histamine H1-receptors by sodium ions and by 2-amino-2-hydroxymethyl-propan-1,3-diol HCl. Br J Pharmacol. 1994 Apr;111(4):1262–1268. doi: 10.1111/j.1476-5381.1994.tb14882.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gosselin R. E., Gosselin R. S. Tachyphylaxis of guinea-pig ileum to histamine and furtrethonium. J Pharmacol Exp Ther. 1973 Feb;184(2):494–505. [PubMed] [Google Scholar]
  17. Harden T. K., Petch L. A., Traynelis S. F., Waldo G. L. Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors. J Biol Chem. 1985 Oct 25;260(24):13060–13066. [PubMed] [Google Scholar]
  18. Hausdorff W. P., Caron M. G., Lefkowitz R. J. Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J. 1990 Aug;4(11):2881–2889. [PubMed] [Google Scholar]
  19. Hertel C., Affolter H., Portenier M., Staehelin M. Desensitized beta-adrenoceptors of C6-glioma cells have distinct binding properties. Naunyn Schmiedebergs Arch Pharmacol. 1984 Nov;328(1):51–55. doi: 10.1007/BF00496106. [DOI] [PubMed] [Google Scholar]
  20. Hertel C., Müller P., Portenier M., Staehelin M. Determination of the desensitization of beta-adrenergic receptors by [3H]CGP-12177. Biochem J. 1983 Dec 15;216(3):669–674. doi: 10.1042/bj2160669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hertel C., Staehelin M., Perkins J. P. Evidence for intravesicular beta-adrenergic receptors in membrane fractions from desensitized cells: binding of the hydrophilic ligand CGP-12177 only in the presence of alamethicin. J Cyclic Nucleotide Protein Phosphor Res. 1983;9(2):119–128. [PubMed] [Google Scholar]
  22. Hill S. J., Young J. M. Characterization of [3H]mepyramine binding to the longitudinal muscle of guinea pig small intestine. Mol Pharmacol. 1981 May;19(3):379–387. [PubMed] [Google Scholar]
  23. Hishinuma S., Uchida M. K. Blockade by the local anaesthetic, tetracaine, of desensitization of Ca-induced Ca release after muscarinic stimulation in smooth muscle. Br J Pharmacol. 1991 Jun;103(2):1393–1398. doi: 10.1111/j.1476-5381.1991.tb09800.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hishinuma S., Uchida M. K. Effects of local anaesthetics on short-term desensitization of guinea-pig taenia caecum to histamine. Br J Pharmacol. 1987 Dec;92(4):733–741. doi: 10.1111/j.1476-5381.1987.tb11377.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hishinuma S., Uchida M. K. Short-term desensitization of guinea-pig taenia caecum induced by carbachol occurs at intracellular Ca stores and that by histamine at H1-receptors. Br J Pharmacol. 1988 Jul;94(3):882–889. doi: 10.1111/j.1476-5381.1988.tb11600.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Horio S., Nakamura S., Ishida Y. Alterations in histamine receptors of guinea-pig ileal smooth muscle produced during agonist-induced desensitization. Br J Pharmacol. 1990 Nov;101(3):587–590. doi: 10.1111/j.1476-5381.1990.tb14125.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Horio S., Shima M., Ueda H., Ishida Y. Temperature-dependence of desensitization induced by acetylcholine and histamine in guinea-pig ileal longitudinal muscle. Br J Pharmacol. 1990 Jul;100(3):636–640. doi: 10.1111/j.1476-5381.1990.tb15859.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hunyady L., Baukal A. J., Balla T., Catt K. J. Independence of type I angiotensin II receptor endocytosis from G protein coupling and signal transduction. J Biol Chem. 1994 Oct 7;269(40):24798–24804. [PubMed] [Google Scholar]
  29. Hunyady L., Merelli F., Baukal A. J., Balla T., Catt K. J. Agonist-induced endocytosis and signal generation in adrenal glomerulosa cells. A potential mechanism for receptor-operated calcium entry. J Biol Chem. 1991 Feb 15;266(5):2783–2788. [PubMed] [Google Scholar]
  30. Kanba S., Kanba K. S., McKinney M., Pfenning M., Abraham R., Nomura S., Enloes L., Mackey S., Richelson E. Desensitization of muscarinic M1 receptors of murine neuroblastoma cells (clone N1E-115) without receptor down-regulation and protein kinase C activity. Biochem Pharmacol. 1990 Sep 1;40(5):1005–1014. doi: 10.1016/0006-2952(90)90486-5. [DOI] [PubMed] [Google Scholar]
  31. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  32. Leeb-Lundberg L. M., Cotecchia S., DeBlasi A., Caron M. G., Lefkowitz R. J. Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of alpha 1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells. J Biol Chem. 1987 Mar 5;262(7):3098–3105. [PubMed] [Google Scholar]
  33. Leurs R., Bast A., Timmerman H. High affinity, saturable [3H]mepyramine binding sites on rat liver plasma membrane do not represent histamine H1-receptors. A warning. Biochem Pharmacol. 1989 Jul 1;38(13):2175–2180. doi: 10.1016/0006-2952(89)90073-7. [DOI] [PubMed] [Google Scholar]
  34. Leurs R., Smit M. J., Bast A., Timmerman H. Different profiles of desensitization dynamics in guinea-pig jejunal longitudinal smooth muscle after stimulation with histamine and methacholine. Br J Pharmacol. 1990 Dec;101(4):881–888. doi: 10.1111/j.1476-5381.1990.tb14175.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Leurs R., Smit M. J., Bast A., Timmerman H. Homologous histamine H1 receptor desensitization results in reduction of H1 receptor agonist efficacy. Eur J Pharmacol. 1991 Apr 24;196(3):319–322. doi: 10.1016/0014-2999(91)90446-w. [DOI] [PubMed] [Google Scholar]
  36. Liu Y. Q., Horio Y., Mizuguchi H., Fujimoto K., Imamura I., Abe Y., Fukui H. Re-examination of [3H]mepyramine binding assay for histamine H1 receptor using quinine. Biochem Biophys Res Commun. 1992 Nov 30;189(1):378–384. doi: 10.1016/0006-291x(92)91569-c. [DOI] [PubMed] [Google Scholar]
  37. Lohse M. J., Benovic J. L., Caron M. G., Lefkowitz R. J. Multiple pathways of rapid beta 2-adrenergic receptor desensitization. Delineation with specific inhibitors. J Biol Chem. 1990 Feb 25;265(6):3202–3211. [PubMed] [Google Scholar]
  38. Maloteaux J. M., Gossuin A., Waterkeyn C., Laduron P. M. Trapping of labelled ligands in intact cells: a pitfall in binding studies. Biochem Pharmacol. 1983 Sep 1;32(17):2543–2548. doi: 10.1016/0006-2952(83)90016-3. [DOI] [PubMed] [Google Scholar]
  39. McCreath G., Hall I. P., Hill S. J. Agonist-induced desensitization of histamine H1 receptor-mediated inositol phospholipid hydrolysis in human umbilical vein endothelial cells. Br J Pharmacol. 1994 Nov;113(3):823–830. doi: 10.1111/j.1476-5381.1994.tb17067.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mitsuhashi M., Payan D. G. Characterization of functional histamine H1 receptors on a cultured smooth muscle cell line. J Cell Physiol. 1988 Mar;134(3):367–375. doi: 10.1002/jcp.1041340307. [DOI] [PubMed] [Google Scholar]
  41. Nakahata N., Harden T. K. Regulation of inositol trisphosphate accumulation by muscarinic cholinergic and H1-histamine receptors on human astrocytoma cells. Differential induction of desensitization by agonists. Biochem J. 1987 Jan 15;241(2):337–344. doi: 10.1042/bj2410337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Quach T. T., Duchemin A. M., Rose C., Schwartz J. C. Specific desensitization of histamine H1 receptor-mediated [3H]glycogen hydrolysis in brain slices. Mol Pharmacol. 1981 Sep;20(2):331–338. [PubMed] [Google Scholar]
  43. Raymond J. R., Albers F. J., Middleton J. P., Lefkowitz R. J., Caron M. G., Obeid L. M., Dennis V. W. 5-HT1A and histamine H1 receptors in HeLa cells stimulate phosphoinositide hydrolysis and phosphate uptake via distinct G protein pools. J Biol Chem. 1991 Jan 5;266(1):372–379. [PubMed] [Google Scholar]
  44. Smit M. J., Bloemers S. M., Leurs R., Tertoolen L. G., Bast A., de Laat S. W., Timmerman H. Short-term desensitization of the histamine H1 receptor in human HeLa cells: involvement of protein kinase C dependent and independent pathways. Br J Pharmacol. 1992 Oct;107(2):448–455. doi: 10.1111/j.1476-5381.1992.tb12766.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Taylor J. E., Richelson E. Desensitization of histamine H1 receptor-mediated cyclic GMP formation in mouse neuroblastoma cells. Mol Pharmacol. 1979 May;15(3):462–471. [PubMed] [Google Scholar]
  46. Treherne J. M., Young J. M. Temperature-dependence of the kinetics of the binding of [3H]-(+)-N-methyl-4-methyldiphenhydramine to the histamine H1-receptor: comparison with the kinetics of [3H]-mepyramine. Br J Pharmacol. 1988 Jul;94(3):811–822. doi: 10.1111/j.1476-5381.1988.tb11592.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Treherne J. M., Young J. M. [3H]-(+)-N-methyl-4-methyldiphenhydramine, a quaternary radioligand for the histamine H1-receptor. Br J Pharmacol. 1988 Jul;94(3):797–810. doi: 10.1111/j.1476-5381.1988.tb11591.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Waldo G. L., Northup J. K., Perkins J. P., Harden T. K. Characterization of an altered membrane form of the beta-adrenergic receptor produced during agonist-induced desensitization. J Biol Chem. 1983 Nov 25;258(22):13900–13908. [PubMed] [Google Scholar]
  49. Wallace R. M., Young J. M. Temperature dependence of the binding of [3H]mepyramine and related compounds to the histamine H1 receptor. Mol Pharmacol. 1983 Jan;23(1):60–66. [PubMed] [Google Scholar]
  50. White M. F., Kahn C. R. The insulin signaling system. J Biol Chem. 1994 Jan 7;269(1):1–4. [PubMed] [Google Scholar]
  51. Wojcikiewicz R. J., Tobin A. B., Nahorski S. R. Muscarinic receptor-mediated inositol 1,4,5-trisphosphate formation in SH-SY5Y neuroblastoma cells is regulated acutely by cytosolic Ca2+ and by rapid desensitization. J Neurochem. 1994 Jul;63(1):177–185. doi: 10.1046/j.1471-4159.1994.63010177.x. [DOI] [PubMed] [Google Scholar]

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