title: AF-DX 384 binding in rabbit cingulate cortex: two site kinetics and section autoradiography.
abstract: Autoradiographic studies of muscarinic receptors are limited by the lack of selective ligands. Inasmuch as AF-DX 384 has a higher affinity for m2 than m4 receptors and pirenzepine (PZ) has a reverse affinity profile, competition between these ligands was used to label m2 receptors in homogenized and sectioned tissue. Rabbit cingulate cortex was used because m2 receptors are expressed by anterior thalamic axons in posterior cingulate cortex (PCC) and this region is easily deafferented with undercut lesions to demonstrate presynaptic binding. Saturation isotherms and Scatchard analysis of [3H]AF-DX 384 binding showed one binding site with a KD of 9 +/- 2.3 nM (mean +/- SEM) and a Bmax of 1405 +/- 146 fmol/mg protein. Competition studies with [3H]AF-DX 384 (2 nM) and 10(-10)-10(-4) M PZ were performed in anterior cingulate cortex (ACC) and PCC. In both regions, the best fit was a two site model for low (BL) and high (BH) affinity binding in which Bmax values were similar (ACC: BL = 535 +/- 62 fmol/mg, BH = 676 +/- 85; PCC: BL = 552 +/- 41; BH = 675 +/- 85). Although affinities for KH were similar in each region (ACC: KH = 4.69 +/- 1.36 nM; PCC: KH = 8.53 +/- 3.69 nM), those for KL were significantly different (ACC: 181 +/- 15.4 nM; PCC: 285 +/- 42; P = .018). Binding of [3H]AF-DX 384 with PZ (150 nM) was best fit with a single site model (KD = 6 +/- 0.01 nM; Bmax = 688 +/- 31 fmol/mg), suggesting that PZ blocks the lower affinity site. (ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Physiology and Pharmacology Bowman Gray School of Medicine Wake Forest University Winston-Salem, North Carolina, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Dopke
authors.forename: K E
authors.initials: KE
authors.lastname: Vrana
authors.forename: L J
authors.initials: LJ
authors.lastname: Vogt
authors.forename: B A
authors.initials: BA
authors.lastname: Vogt
mesh_terms: D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D006179:Gyrus Cinguli / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008297:Male; D010276:Parasympatholytics / Q000378:metabolism*; D010890:Pirenzepine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D011817:Rabbits; D011976:Receptors, Muscarinic / Q000378:metabolism
chemical_list: D008024:Ligands; D010276:Parasympatholytics; D011976:Receptors, Muscarinic; C065959:AFDX 384; D010890:Pirenzepine