Further evidence for the existence of two forms of alpha 2B-adrenoceptors in rat.
Analysis of saturation isotherms of the novel alpha 2-adrenoceptor antagonist radioligand [3H]-MK 912 revealed that the ligand labelled a homogenous population of alpha 2B-adrenoceptors in the neonatal rat lung with a Kd of 0.77 nM and a Bmax of 231 fmol/mg protein. In rat kidney, combined saturation and competition experiments, using [3H]-MK 912 and the alpha 2A-adrenoceptor selective drug guanfacine, revealed that approximately 81% of the sites labelled by [3H]-MK 912 were alpha 2B-adrenoceptors and approximately 19% alpha 2A-adrenoceptors; the Kds of [3H]-MK 912 being 1.1 and 2.0 nM and the Bmax 134 and 33 fmol/mg protein, respectively. The kidney alpha 2B-adrenoceptors were studied separately by using approximately 1.5 nM [3H]-MK 912 in the presence of 0.32 microM guanfacine, the latter which blocked ligand binding to alpha 2A-adrenoceptors completely. Analysis of drug competition curves obtained during these conditions revealed that 18 out of 20 different agonists and antagonists yielded steep and uniphasic competition curves which modelled best into one site fits. However, both guanoxabenz and LT 11 appeared to inhibit [3H]-MK 912 binding at two sites; the Kds of guanoxabenz differing approximately 120-fold and that of LT 11 differing approximately 35-fold for the two sites. Moreover, the addition of mutual fixed concentrations of either 20 microM guanoxabenz or 20 microM LT 11 completely prevented the binding of the other compound to its high affinity site, indicating that both compounds labelled the same site with the high affinity. The analysis indicated that 29% of the sites were of high and 71% of low affinity. However, in the neonatal rat lung guanoxabenz and LT 11 (as well as 15 other compounds) yielded competition curves which modelled only into one site fits. The Kds obtained in the lung correlated well with the Kds obtained in the kidney for alpha 2B-adrenoceptors; for guanoxabenz and LT 11 the values from the lung were close to those determined in the kidney for the low affinity site for guanoxabenz and LT 11. Moreover, when the rat RNG alpha 2B-adrenoceptor was expressed in COS-7 cells and its binding properties tested using [3H]-MK 912 binding, guanoxabenz, LT 11 as well as a number of other drugs inhibited the ligand binding at a single alpha 2-adrenoceptor site; the drug Kds being practically the same as those found for the neonatal rat lung. It is suggested that rat alpha 2B-adrenoceptors may exist in two forms: alpha 2B1 and alpha 2B2.(ABSTRACT TRUNCATED AT 400 WORDS)
Xia
Y
Y
Department of Pharmacology, Umeå University, Sweden.
Uhlén
S
S
Chhajlani
V
V
Lien
E J
EJ
Wikberg
J E
JE
D000317:Adrenergic alpha-Antagonists / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D016316:Guanfacine / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008168:Lung / Q000378:metabolism; D008297:Male; D011807:Quinolizines / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*
D000317:Adrenergic alpha-Antagonists; D008024:Ligands; D011807:Quinolizines; D011942:Receptors, Adrenergic, alpha; C054290:L 657743; D016316:Guanfacine