Skip to main content
NCBI home page
Springer Nature - PMC COVID-19 Collection logoLink to Springer Nature - PMC COVID-19 Collection
. 1993;347(3):280–288. doi: 10.1007/BF00167446

Evidence for the existence of two forms of α2A-adrenoceptors in the rat

Staffan Uhlén 1,✉, Yun Xial 1, Vijay Chhajlanil 1, Eric J Lien 3, Jarl E S Wikberg 1
PMCID: PMC7087569  PMID: 8097566

Summary

The α2A-adrenoceptors in rat spleen, kidney, spinal cord and cerebral cortex were studied using [3H]-RX821002 radioligand binding. In the spleen, spinal cord and cerebral cortex, the ligand bound to saturable sites with a K d of about 1 nmol/l and capacities of 134, 240 and 290 fmol/mg protein, respectively. Computer modelling competition curves for 39 drugs, including those for α2A-, α2B- or α2C-adrenoceptor selective drugs, indicated that the sites labelled by [3H]-RX821002 in the spleen consisted of a single population of α2A-adrenoceptors. However, the competition curves for guanoxabenz were definitely biphasic and resolved into two site fits, indicating that guanoxabenz was binding to both high affinity (K d = 35 nmol/1) and low affinity (K d = 8900 nmol/1) α2A-adrenoceptor sites in the proportions 57% and 43%, respectively. The K d Sfor a number of α2-adrenoceptor subtype selective drugs, measured in competition with [3H]-RX821002 in cerebral cortex and spinal cord, were highly correlated with those obtained in the spleen indicating their α2A-adrenoceptor nature. However, by contrast to the results with the spleen, the guanoxabenz competition curves for the spinal cord and cerebral cortex were monophasic and resolved only into one site fits, the K d of guanoxabenz being about 4000 nmol/l for both tissues. Drug K d Sfor kidney α2A-adrenoceptors were also determined using [3H]-RX821002. For nearly all drugs tested, the K d Swere highly correlated with those found for the α2A-adrenoceptors in the other rat tissues. However, for guanoxabenz, the data indicated that it competed with [3H]-RX821002 at a single α2A-adrenoceptor site with a K d of 39 nmol/1. When the rat α2A-adrenoceptor gene RG20 was transiently expressed in COS-7 cells and its ligand binding properties probed using [3H]-RX821002, the drug K d Sobtained were also highly correlated with those found for the α2A-adrenoceptors in the spleen, cerebral cortex, spinal cord and kidney of the rat. For the RG20 encoded receptor, the guanoxabenz competition curves were steep and monophasic and modelled best into one site fits, with the Kd of guanoxabenz being 5200 nmol/1.

It is suggested that guanoxabenz can differentiate between two forms of α2A-adrenoceptors in the rat: α2A1 and α2A2. The α2A1-form is present in the spleen and kidney where it shows a high apparent affinity for guanoxabenz. The α2A2-form shows a low apparent affinity for guanoxabenz and is present in the spleen, cerebal cortex and spinal cord. The α2A2-form of the rat α2-adrenoceptor appears to be encoded by the RG20 gene. The α2A, and α2A2-adrenoceptor forms do not represent high and low affinity receptor forms for agonists because assays included EDTA, Gpp(NH)p and Na+, which eliminated the high affinity receptors for agonists.

Key words: α2A-Adrenoceptor forms, [3H]-RX821002 ligand binding, Rat tissues, Expressed RG20 α2-adrenoceptor, Guanoxabenz

References

  1. Bylund DB. Heterogeneity of alpha-2-adrenergic receptors. Pharmacol Biochem Behav. 1985;22:835–843. doi: 10.1016/0091-3057(85)90536-2. [DOI] [PubMed] [Google Scholar]
  2. Bylund DB. Subtypes of α2-adrenoceptors: Pharmacological and molecular biological evidence converge. Trends Pharmacol Sci. 1988;9:356–361. doi: 10.1016/0165-6147(88)90254-4. [DOI] [PubMed] [Google Scholar]
  3. Bylund DB. Subtypes of α1- and α2-adrenergic receptors. FASEB J. 1992;6:832–839. doi: 10.1096/fasebj.6.3.1346768. [DOI] [PubMed] [Google Scholar]
  4. Bylund DB, Ray-Prenger C, Murphy TJ. Alpha-2A and alpha-2B adrenergic receptor subtypes: antagonist binding in tissues and cell lines containing only one subtype. J Pharmacol Exp Ther. 1988;245:600–607. [PubMed] [Google Scholar]
  5. Bylund DB, Blaxall HS, Murphy TJ, Simmoneaux V. Pharmacological evidence for alpha-2C and alpha-2D adrenergic receptor subtypes. In: Szabadi E, Bradshaw CM, editors. Adrenoceptors: Structure, mechanisms, function. Basel: Birkhauser; 1991. pp. 27–36. [Google Scholar]
  6. Chalberg SC, Duda T, Rhine JA, Sharma RK. Molecular cloning, sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain. Mol Cell Biochem. 1990;97:161–172. doi: 10.1007/BF00221058. [DOI] [PubMed] [Google Scholar]
  7. Harrison JK, Pearson WR, Lynch KR. Molecular characterization of α1- and α2-adrenoceptors. Trends Pharmacol Sci. 1991;12:62–67. doi: 10.1016/0165-6147(91)90499-i. [DOI] [PubMed] [Google Scholar]
  8. Harrison JK, D'Angelo DD, Zeng D, Lynch KR. Pharmacological characterization of rat α2-adrenergic receptors. Mol Pharmacol. 1991;40:407–412. [PubMed] [Google Scholar]
  9. Horstman DA, Brandon S, Wilson AL, Guyer CA, Cragoe EJ, Jr, Limbird LE. An aspartate conserved among G-protein receptors confers allosteric regulation of α2-adrenergic receptors by sodium. J Biol Chem. 1990;265:21590–21595. [PubMed] [Google Scholar]
  10. Kobilka BK, Matsui H, Kobilka TS, Yang-Feng TL, Francke U, Caron MG, Lefkowitz RJ, Regan JW. Cloning, sequencing, and expression of the gene coding for the human platelet α2-adrenergic receptor. Science. 1987;238:650–656. doi: 10.1126/science.2823383. [DOI] [PubMed] [Google Scholar]
  11. Langin D, Paris H, Lafontan M. Binding of [3H]idazoxan and its methoxy derivative [3]HIRX821002 in human fat cells: [3H]idazoxan but not [3H]RX821002 labels additional non-α2-adrenergic binding sites. Mol Pharmacol. 1990;37:876–885. [PubMed] [Google Scholar]
  12. Lanier SM, Downing S, Duzic E, Homey CJ. Isolation of rat genomic clones encoding subtypes of the α2-adrenergic receptor. Identification of a unique receptor subtype. J Biol Chem. 1991;266:10470–10478. [PubMed] [Google Scholar]
  13. Link R, Daunt D, Barsh G, Chruscinski A, Kobilka B. Cloning of two mouse genes encoding α2-adrenergic receptor subtypes and identification of a single amino acid in the mouse α2-C10 homolog responsible for an interspecies variation in antagonist binding. Mol Pharmacol. 1992;42:16–27. [PubMed] [Google Scholar]
  14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275. [PubMed] [Google Scholar]
  15. Michel AD, Loury DN, Whiting RL. Differences between the alpha 2-adrenoceptor in rat submaxillary gland and the alpha 2A- and alpha 2B-adrenoceptor subtypes. Br J Pharmacol. 1989;98:890–897. doi: 10.1111/j.1476-5381.1989.tb14618.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Michel T, Hoffman BB, Lefkowitz RJ. Differential regulation of the α2-adrenergic receptor by Na+ and guanine nucleotides. Nature. 1980;288:709–711. doi: 10.1038/288709a0. [DOI] [PubMed] [Google Scholar]
  17. Snavely MD, Insel PA. Characterization of α-adrenergic subtypes in the rat renal cortex. Differential regulation of αl- and α2-adrenergic receptors by guanyl nucleotides and Na+ Mol Pharmacol. 1982;22:532–546. [PubMed] [Google Scholar]
  18. Tai AW, Lien EJ, Lai MMC, Khwaja TA. Novel N-hydroxyguanidine derivatives as anticancer and antiviral agents. J Med Chem. 1984;27:236–238. doi: 10.1021/jm00368a024. [DOI] [PubMed] [Google Scholar]
  19. Tang A, Lien EJ, Lai MMC. Optimization of the Schiff bases of N-hydroxy-N′-aminoguanidine as anticancer and antiviral agents. J Med Chem. 1985;28:1103–1106. doi: 10.1021/jm00146a022. [DOI] [PubMed] [Google Scholar]
  20. Uhlén S, Wikberg JES. Delineation of three pharmacological subtypes of α2-adrenoceptors in the kidney. Br J Pharmacol. 1991;104:657–664. doi: 10.1111/j.1476-5381.1991.tb12485.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Uhlén S, Wikberg JES. Rat spinal cord α2-adrenoceptors are of the α2A-subtype: Comparison with the α2A- and α2B-adrenoceptors in rat spleen, cerebral cortex and kidney using 3H-RX821002 ligand binding. Pharmacol Toxicol. 1991;69:341–350. doi: 10.1111/j.1600-0773.1991.tb01308.x. [DOI] [PubMed] [Google Scholar]
  22. Uhlén S, Wikberg JES. Delineation of rat kidney α2A- and α2B-adrenoceptors with [3H]RX821002 radioligand binding: computer modelling reveals that guanfacine is an α2A-selective compound. Eur J Pharmacol. 1991;202:235–243. doi: 10.1016/0014-2999(91)90299-6. [DOI] [PubMed] [Google Scholar]
  23. Uhlén S, Xia Y, Chhajlani V, Felder CC, Wikberg JES. [3H]MK912 binding delineates two α2-adrenoceptor subtypes in rat CNS one of which is identical with the cloned pA2d α2-adrenoceptor. Br J Pharmacol. 1992;106:986–995. doi: 10.1111/j.1476-5381.1992.tb14446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Voigt MM, McCune SK, Kanterman RY, Felder CC. The rat α2-C4 adrenergic receptor gene encodes a novel pharmacological subtype. FEBS Lett. 1991;278:45–50. doi: 10.1016/0014-5793(91)80080-m. [DOI] [PubMed] [Google Scholar]
  25. Wang P-H, Keck JG, Lien EJ, Lai MMC. Design, synthesis testing, and quantitative structure-activity relationship analysis of substituted salicylaldehyde Schiff bases of 1-amino-3-hydroxyguanidine tosylate as new antiviral agents against coronavirus. J Med Chem. 1990;22:608–614. doi: 10.1021/jm00164a023. [DOI] [PubMed] [Google Scholar]
  26. Wikberg JES. High affinity binding of idazoxan to a non-catecholaminergic binding site in the central nervous system: description of a putative idazoxan-receptor. Pharmacol Toxicol. 1989;64:152–155. doi: 10.1111/j.1600-0773.1989.tb00620.x. [DOI] [PubMed] [Google Scholar]
  27. Wikberg JES, Uhlén S. Further characterization of the guinea pig cerebral cortex idazoxan-receptor. Solubilization, distinction from the imidazole-site and demonstration of cirazoline as an idazoxan-receptor selective drug. J Neurochem. 1990;55:192–203. doi: 10.1111/j.1471-4159.1990.tb08838.x. [DOI] [PubMed] [Google Scholar]
  28. Wikberg JES, Uhlén S, Chhajlani V. Medetomidine stereoisomers delineate two closely related subtypes of idazoxan (imidazoline) I-receptors in the guinea pig. Eur J Pharmacol. 1991;193:335–340. doi: 10.1016/0014-2999(91)90148-j. [DOI] [PubMed] [Google Scholar]
  29. Xia Y, Uhlén S, Lien EJ, Wikberg JES (1993) Further evidence for the existence of two forms of α2B-adrenoceptors in the rat. Pharmacol Toxicol (in press) [DOI] [PubMed]
  30. Zeng DW, Harrison JK, D Angelo DD, Barber CM, Tucker AL, Lu Z, Lynch KR. Molecular characterization of a rat α2B-adrenergic receptor. Proc Natl Acad Sci USA. 1990;87:3102–3106. doi: 10.1073/pnas.87.8.3102. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Naunyn-Schmiedeberg's Archives of Pharmacology are provided here courtesy of Nature Publishing Group

RESOURCES